Tuning the bioactivity of tensioactive deoxy glycosides to structure: Antibacterial activity versus selective cholinesterase inhibition rationalized by molecular docking

Article abstract of DOI:10.1002/ejoc.201201520

New octyl/dodecyl 2,6-dideoxy-D-arabino-hexopyranosides have been synthesized by a simple but efficient methodology based on the reaction of glycals with alcohols catalysed by triphenylphosphane hydrobromide, deprotection, regioselective tosylation and reduction. Their surface-active properties were evaluated in terms of adsorption and aggregation parameters and compared with those of 2-deoxy-D-glycosides and 2,6-dideoxy-L-glycosides. Deoxygenation at the 6-position led to a decrease in the critical micelle concentration, and an increase in the adsorption efficiency (pC₂₀) promoting aggregation more efficiently than adsorption. With regard to the antibacterial activity, dodecyl 2,6-dideoxy-α-L-arabino-hexopyranoside was the most active compound towards Bacillus anthracis (MIC 25 μM), whereas its enantiomer exhibited a MIC value of 50 μM. Both 2,6-dideoxy glycosides were active towards Bacillus cereus, Bacillus subtilis, Enterococcus faecalis and Listeria monocytogenes. In contrast, none of the 2-deoxy glycosides was significantly active. These results and the data on surface activity suggest that aggregation is a key issue for antimicrobial activity. Beyond infection, Alzheimer's disease also threatens elderly populations. In the search for butyrylcholinesterase (BChE) selective inhibition, 2-deoxy glycosides were screened in vitro by using Ellman's assay. Octyl 2-deoxy-α-D-glycoside was found to be a BChE selective inhibitor promoting competitive inhibition. Docking studies supported these results as they pinpoint the importance of the primary OH group in stabilizing the BChE inhibitor complex. A size-exclusion mechanism for inhibition has been proposed based on the fact that acetylcholinesterase (AChE) exhibits several bulky residues that hinder access to the active-site cavity. This work shows how the deoxygenation pattern, configuration and functionality of the anomeric centre can tune physical and surface properties as well as the bioactivity of these multifunctional and stereochemically rich molecules. Alkyl 2-deoxy-arabino-hexopyranosides have been synthesized and their surface-active and biological properties tuned by their deoxygenation pattern and anomeric configuration. Dodecyl 2,6-dideoxy-α- glycosides showed the highest antibacterial activity, whereas 2-deoxy-α-glycosides exhibited selective BChE inhibition, rationalized by molecular docking. Copyright

Full text of DOI:10.1002/ejoc.201201520

FULL PAPER
DOI: 10.1002/ejoc.201201520
Tuning the Bioactivity of Tensioactive Deoxy Glycosides to Structure:
Antibacterial Activity Versus Selective Cholinesterase Inhibition Rationalized
by Molecular Docking
Alice Martins,[a] Maria S. Santos,[a] Catarina Dias,[a] Patrícia Serra,[a] Vasco Cachatra,[a]
João Pais,[a] João Caio,[a] Vítor H. Teixeira,[a] Miguel Machuqueiro,[a] Marta S. Silva,[a]
Ana Pelerito,[b] Jorge Justino,[c] Margarida Goulart,[c] Filipa V. Silva,[c] and
Amélia P. Rauter*[a]
Dedicated to the centenary of the Portuguese Chemical Society on the occasion of the
6th Spanish Portuguese Japanese Organic Chemistry Symposium
Keywords: Synthetic methods / Medicinal chemistry / Glycosides / Surfactants / Biological activity / Molecular docking
New octyl/dodecyl 2,6-dideoxy-
D
-arabino-hexopyranosides
and the data on surface activity suggest that aggregation is
a key issue for antimicrobial activity. Beyond infection, Alz-
heimer’s disease also threatens elderly populations. In the se-
arch for butyrylcholinesterase (BChE) selective inhibition, 2-
deoxy glycosides were screened in vitro by using Ellman’s
have been synthesized by a simple but efficient methodology
based on the reaction of glycals with alcohols catalysed by
triphenylphosphane hydrobromide, deprotection, regioselec-
tive tosylation and reduction. Their surface-active properties
were evaluated in terms of adsorption and aggregation pa-
assay. Octyl 2-deoxy-α-D-glycoside was found to be a BChE
rameters and compared with those of 2-deoxy-
D
-glycosides
selective inhibitor promoting competitive inhibition. Docking
studies supported these results as they pinpoint the impor-
tance of the primary OH group in stabilizing the BChE inhib-
itor complex. A size-exclusion mechanism for inhibition has
been proposed based on the fact that acetylcholinesterase
(AChE) exhibits several bulky residues that hinder access to
the active-site cavity. This work shows how the deoxygen-
ation pattern, configuration and functionality of the anomeric
centre can tune physical and surface properties as well as
the bioactivity of these multifunctional and stereochemically
rich molecules.
and 2,6-dideoxy- -glycosides. Deoxygenation at the 6-posi-
L
tion led to a decrease in the critical micelle concentration,
and an increase in the adsorption efficiency (pC20) promoting
aggregation more efficiently than adsorption. With regard to
the antibacterial activity, dodecyl 2,6-dideoxy-α-
hexopyranoside was the most active compound towards Ba-
cillus anthracis (MIC 25 μ ), whereas its enantiomer exhib-
ited a MIC value of 50 μ . Both 2,6-dideoxy glycosides were
L-arabino-
M
M
active towards Bacillus cereus, Bacillus subtilis, Enterococcus
faecalis and Listeria monocytogenes. In contrast, none of the
2-deoxy glycosides was significantly active. These results
tions in the nature of the sugar and hydrocarbon tail length
determine the self-organization properties and therefore ap-
plications of the resulting surfactant.[1] These molecules are
also known for their biocompatibility, low toxicity and fast
biodegradation, which make them very attractive for indus-
trial applications.[2]
The active site of many pharmacologically active amphi-
philic molecules is frequently the plasma membrane. When
the target is intracellular, the interaction with this first bar-
rier may determine their activity. Thus, the interaction of
potential drugs with membranes plays a fundamental role
by either blocking permeation, inducing solubilization[3] or
modifying the membrane structure. In addition, membrane
protein conformation disruption alters important functions
such as transport and energy generation.[4] Surface-active
drugs with very different chemical structures are known to
Introduction
Sugar-based surfactants are gaining increasing attention
due to their biological and medicinal applications. Varia-
[a] Centro de Química e Bioquímica, Faculdade de Ciências da
Universidade de Lisboa,
Ed. C8, Piso 5, Campo Grande, 1749-016 Lisboa, Portugal
Fax: +351-21-7500088
E-mail: aprauter@fc.ul.pt
Homepage: http://carbohydrate.cqb.fc.ul.pt/Profiles/
aprauter.pdf
[b] Instituto Nacional de Saúde Doutor Ricardo Jorge,
Av. Padre Cruz, 1649-016 Lisboa, Portugal
[c] Escola Superior Agrária de Santarém, Instituto Politécnico de
Santarém, Quinta do Galinheiro,
2001-904 Santarém, Portugal
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201201520.
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Tuning the Bioactivity of Deoxy Glycosides to Structure
self-associate and bind to membranes, acting in a detergent- of the d series by a simple but efficient methodology and
like manner, accumulating and affecting the organization evaluated their surface-active properties and antimicrobial
and thermotropic properties of lipid bilayers.[3,5,6] Tradi- activities. Furthermore, in vitro screening for AChE and
tional hard cationic surfactants exhibit high antimicrobial BChE inhibition by alkyl 2-deoxy and alkyl 2,6-dideoxy
activity due to their membrane disruptive nature, but can glycosides was carried out and docking studies were per-
damage mammalian cells upon long exposure, whereas neu- formed to rationalize the selective inhibition observed and
tral surfactants are known for their soft antimicrobial prop- to elucidate the stereoselectivity and binding properties of
erties.[7]
Our previous studies on alkyl deoxy-hexopyranosides of
the active molecules.
The results reported herein clearly demonstrate that sim-
the d and l series in their α- and β-anomeric configura- ple and easily achieved structural modifications to the gly-
tions[8,9] have shown that high surface activity is a pre-req- con enable tuning of the glycoside bioactivity, thus enhanc-
uisite for their antimicrobial activity, but the specificity ap- ing the importance of these multifunctional and stereo-
pears to be linked to the anomeric configuration of the chemically rich molecules.
sugar and to the deoxygenation pattern.[8,9] They are active
towards Listeria monocytogenes and Enterococcus faecalis,
which cause life-threatening infections in humans, particu-
Results and Discussion
larly affecting older individuals who are more susceptible
to infections, especially in hospitals, where high levels of
antibiotic resistance are found. A potent activity towards
Bacillus spp. was also found, in particular towards Bacillus
cereus, a pathogenic microbe, the genome of which is very
Synthesis
The 2-deoxy glycosides were synthesized in good yields
similar to that of Bacillus anthracis,[10] a well-known bio- by the reaction of glycals with fatty alcohols in the presence
terrorism agent responsible for anthrax infections in live- of triphenylphosphane hydrobromide (TPHB).[9,18] With
stock and humans. Recently, 2,4-diaminopyrimidines were this catalyst, the Ferrier products, which result from reac-
reported to inhibit this microbe.[11] As a result of the re- tion of acetylated glycals with alcohols catalysed by Lewis
ported microbial resistance to this family of antibiotics and acids, are absent or can be isolated as secondary products in
to many other available therapeutics,[12] the synthesis of new very low yields. Zemplén deacetylation afforded the target
antimicrobial agents with new mechanisms of action for the glycosides in almost quantitative yields. Because the 6-de-
effective treatment of infectious diseases is mandatory. oxy glycal of the d series was not commercially available,
Herein, we present the first results concerning the anti- we synthesized compounds 6–9 starting from the glycal
microbial activity of a new family of antibiotics based on 3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxy-d-arabino-hex-1-en-
alkyl deoxy glycosides synthesized by our group[13] towards itol (1; Scheme 1). Tosylation at the primary position was
three strains of B. anthracis.
easily accomplished with tosyl chloride in pyridine and
Other age-associated pathologies include neurodegener- dichloromethane at 0 °C in yields ranging from 84 to 93%
ative disorders, namely Alzheimer’s disease (AD), which, it for compounds 10–13. The tosylation of only one OH
is estimated, will affect 70 million people in 2050 if the lack group was confirmed by NMR spectroscopy due to the
of an efficient treatment persists.[14] Its treatment is pres- presence of characteristic doublets of the aromatic protons
ently based on acetylcholinesterase (AChE) or AChE and at δ = 7.80–7.33 ppm and of the singlet at δ = 2.43–
butyrylcholinesterase (BChE) dual inhibitors, and is effec- 2.44 ppm for the methyl group, which integrate for a single
tive only in the early-to-middle stages of the disease. The tosyl group in each compound. Moreover, the displacement
activity of AChE in the brains of AD patients decreases of the chemical shifts of 6a-H and 6b-H to lower fields con-
whereas that of BChE gradually increases up to 40–90%.[15] firmed the selective substitution at 6-OH. Reduction of the
Moreover, selective BChE inhibition results in a concomi- tosylated compounds was first attempted with lithium tri-
tant increase in brain acetylcholine levels and a decrease in ethylborohydride (super-hydride), but the single product
Alzheimer’s β-amyloid in rodents.[16] However, the impact obtained resulted from intramolecular cyclization as a re-
of BChE inhibition on cognitive dysfunction remains to be sult of SN2 displacement of the tosyloxy group by the 3-
determined.[17] Selective BChE inhibitors have been consid- alkoxy group. The reduction of the tosyloxy group suc-
ered an attractive therapy for AD patients but none of them ceeded with lithium aluminium hydride in THF to afford
has reached the market yet. Hence, the search for new mo- the 2,6-dideoxy compounds 14–17 in yields of 70–79%,
lecular entities that exhibit this activity remains a challenge confirmed by the absence of signals corresponding to the
and our research program on healthy ageing has involved tosyl group and the presence of chemical shifts for 6-CH3
the screening of these new compounds for this activity.
at δ = 18.0 ppm (carbon resonance). Although conven-
The results obtained so far for the alkyl 2-deoxy glycos- tional, this is an efficient approach that leads to molecules
ides of the d series and for the 2,6-dideoxy analogues of the with the 2,6-dideoxy pattern in a simple, high-yielding and
l series suggest that this family of compounds should be reproducible manner. The 2,6-dideoxy l series glycosides
further explored and that, in particular, the study of the 18–21 (Scheme 2) were synthesized starting from the com-
2,6-dideoxy analogues of the d series is imperative. In this mercially available 6-deoxy-l-glycal as described pre-
work we have synthesized new alkyl 2,6-dideoxy glycosides viously.[8]
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A. P. Rauter et al.
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Scheme 2. Structure of compounds 18–21 synthesized according to
ref.[8]
Surface Activity
The dependence of the surface tension, γ, on the mo-
lality, m, of the octyl and dodecyl 2,6-dideoxy-α-d-arabino-
hexopyranosides (14 and 16, respectively) was evaluated.
Measurements were also taken for the dodecyl 2,6-dideoxy-
β-d-arabino-hexopyranoside (17), but its very low solubility
led to erroneous measurements for m Ն 1ϫ10–5 mol/kg.
The data obtained from independent batches and dilutions
of these compounds are in good agreement, which recon-
firms the purity of the samples. Furthermore, no minimum
near the CMC was observed in any of the plots (Figure 1),
which indicates the absence of hydrophobic impurities in
the synthesized and purified alkyl 2,6-dideoxy-d-arabino-
hexopyranosides.[19]
Both plots show an identical pattern with three sections
(Figure 1), two of them below the critical micelle concentra-
tion (CMC) and a third one above the CMC. In this last
section the molality is almost independent of the surface
tension. The presence of a break between the first two sec-
tions (47 Ͻ γ Ͻ 40 mN/m) below the CMC indicates the
formation of pre-micellar aggregates, a feature previously
observed in our studies on alkyl 2-deoxy-d- and 2,6-dide-
oxy-l-arabino-hexopyranosides[8,9] as well as with commer-
cial alkyl polyglucosides.[19–21]
The moduli of the pre-micellar slopes and surface excess
obtained from the Gibbs plots (Figure 2) both increase with
alkyl chain length, as expected from the increased hydro-
phobicity of the compounds. Post-micellar slopes for the
octyl derivative 14 are more negative than those of the do-
Scheme 1. Synthesis of compounds 2–17. Reagents and conditions:
i) 0.1 equiv. TPHB, 2 equiv. ROH, anhydrous CH2Cl2, room temp.,
12 h; 69% for 2, 15% for 3, 83% for 4, 12% for 5; ii) a. MeONa,
MeOH, pH 9, 45 min; b. Amberlite IR 120; 97% for 6, 98% for 7,
95% for 8, 97% for 9; c. 2 equiv. TsCl, CH2Cl2/Py (1:1), 0 °C to
room temp., 2 h; 88% for 10, 84% for 11, 93% for 12, 91% for 13;
iii) 3 equiv. LiAlH4, THF, 0 °C to room temp., 72 h; 70% for 14,
72% for 15, 79% for 16, 75% for 17.
Figure 1. Dependence of surface tension on the concentration of aqueous solutions of octyl 2,6-dideoxy-α-d-arabino-hexopyranoside (14)
and dodecyl 2,6-dideoxy-α-d-arabino-hexopyranoside (16) at 35 °C.
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Tuning the Bioactivity of Deoxy Glycosides to Structure
Figure 2. Gibbs plots for aqueous solutions of octyl 2,6-dideoxy-α-d-arabino-hexopyranoside (14) and dodecyl 2,6-dideoxy-α-d-arabino-
hexopyranoside (16) at 35 °C.
decyl derivative, which suggests a less efficient packing at Table 1. Adsorption properties of alkyl deoxy glycosides at the air/
water interface.[a]
the air/water interface and indicates the formation of micel-
lar aggregates with an increased degree of polydispersity.
The surface activity parameters for compounds 14 and
16 were compared with the corresponding data for their
enantiomers 18 and 20, respectively, as well as with data
previously obtained for the 2-deoxy glycosides. Compounds
19 and 21 presented a very low solubility, as observed for
compound 17, and were not tested. The data previously
published[8,9] were refined with a new set of data obtained
for new batches of 18 and 20, evaluated with an automatic
tensiometer instead of the manual Kruss K8 previously
used. Recalculated values for these enantiomers over a
larger concentration range are summarized in Table 1. The
results demonstrate that deoxygenation of the glycoside
head-groups promotes aggregation, leading to a decrease in
the CMC and an increase in the adsorption efficiency
(pC20). Therefore the CMC/C20 ratio was calculated to
screen for the role of structural microenvironmental factors
on both these parameters. The ratio decreases with chain
length for 2-deoxy- and 2,6-dideoxy glycopyranosides of the
l and d series, which indicates that increasing the alkyl
chain promotes aggregation more efficiently than adsorp-
tion. The CMC/C20 ratio decreases more abruptly for the d
series dideoxy derivatives than for 2-deoxy glycosides,
which suggests that removal of the hydroxy group at the 6-
position is crucial to increase aggregation.
The remarkable difference in the magnifying ratio CMC/
C20 of the octyl dideoxy glycosides may result from the
polydisperse nature of these aggregates. A longer alkyl
chain promotes stronger attractive chain interactions and
facilitates molecular aggregation. Although the measured
CMC for dodecyl dideoxy α-d-glycoside is lower than that
[a] am: molecular area on a saturated liquid/air interface.
of its enantiomer, the surface excesses are virtually identical
within their uncertainties. This unexpected behaviour was ences are within the CMC/C20 uncertainties of the dodecyl
verified with different batches of the l enantiomer 20. The glycosides.
decrease observed in the CMC/C20 ratio on going from oc-
The removal of the hydroxy group from the 6-position
tyl to dodecyl dideoxy glycosides of the l series is less than generates other remarkable changes in the physical (ther-
that for their enantiomeric glycosides. This suggests a better motropic) properties and an inversion in the solubility. De-
packing efficiency for the former dideoxy glycosides than oxygenation at only the 2-position leads to direct transitions
for the dideoxy-d-glycopyranosides. However, these differ- from the solid crystalline state to the isotropic liquid melts
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A. P. Rauter et al.
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and to a decrease in the melting points, as observed for Antibacterial Activity
the octyl and dodecyl 2-deoxy-d-arabino-hexopyranosides
The synthesized compounds were evaluated for their an-
timicrobial activity first by the paper disk diffusion method.
The results are expressed as the average diameter of the
inhibition zone detected in three replicates (Table 3).
The minimal inhibitory concentrations (MIC) were de-
termined for those compounds that showed considerable
activity in the preliminary paper disk assays and the results
are presented in Table 3. The dodecyl α-d-glycoside 16
shows the highest diameter of inhibition towards Bacillus
cereus of all the compounds tested, even higher than that
of the reference antibiotic chloramphenicol at the same
concentration. This compound also promoted significant
growth inhibition of Bacillus subtilis, Enterococcus faecalis
and Listeria monocytogenes, with inhibition diameters vary-
ing from 17Ϯ7 to 28Ϯ12 mm. Its stereoisomer with all the
stereogenic centres bearing an inverted configuration, the
dodecyl α-l-glycoside 20, also showed significant activity
towards the Bacillus species, particularly B. cereus. How-
ever, the β anomers 17 and 21 were completely inactive,
which suggests that the anomeric configuration plays an im-
portant role in the antimicrobial activity towards the Bacil-
lus species.
previously studied[8] (Table 2). Nevertheless, the solubility
pattern of d-glucosides is retained, namely the β anomers
are more soluble than the corresponding α anomers.[8,19,20]
However, upon additional deoxygenation at the 6-position,
a small decrease in the melting point of both β anomers
and an abrupt decrease for both α anomers is observed and,
concurrently, an inversion in the solubility pattern is also
detected.
Table 2. Transition temperatures of alkyl glucosides to isotropic li-
quids.
Compound
Transition temp. [°C]
α anomer β anomer
Octyl 2,6-dideoxy-d-arabino-hexopyranoside
Dodecyl 2,6-dideoxy-d-arabino-hexopyrano-
side
Octyl 2,6-dideoxy-l-arabino-hexopyranoside
Dodecyl 2,6-dideoxy-l-arabino-hexopyrano-
side
Octyl 2-deoxy-d-arabino-hexopyranoside
Dodecyl 2-deoxy-d-arabino-hexopyranoside
Octyl d-glucopyranoside
Ͻ25
36
80
97
Ͻ25
36
68[9]
82[9]
107[8]
85.5[8]
102.3[8]
104.5[22]
99[23]
114.8[8]
115[22]
,
Decyl d-glucopyranoside
136[22]
148[22]
140[22]
137[23]
146[22]
,
In spite of the low solubility of the α-d-glycoside 16, the
local concentration on the Bacillus membrane may be sig-
nificant in view of the high value of pC20 determined for
this compound. The presence of the dodecyl chain is also
Dodecyl d-glucopyranoside
These findings suggest that the thermotropic behaviour determinant of the bioactivity exhibited by the 2,6-dideoxy
is dictated by intermolecular hydrogen bonding of the head- glycosides of both the d and l series.
group and tuned by hydrophobic interactions in both gluco-
None of the synthesized compounds at the tested con-
pyranosides[22,23] and 2-deoxy-arabino-hexopyranosides.[8] centrations revealed significant activity towards Salmonella
Further deoxygenation at the 6-position significantly re- enteritidis, Pseudomonas aeruginosa or Escherichia coli.
duces the hydrogen bonding and the melting point and sol- However, the dodecyl 2-deoxy-β-d-glycoside 9 showed sig-
ubility become processes ruled by hydrophobic interac- nificant activity towards Enterococcus faecalis. The α ano-
tions.[24]
mers 6 and 8 were inactive towards all the microbes
The apparent inconsistency in the previously reported tested.[8]
melting point of 20 (34 °C)[9] was verified by evaluating the
The antibacterial activities of this new family of antibiot-
melting point of the newly synthesized compound. A dried ics towards B. anthracis are reported for the first time. The
sample of 20 revealed time-dependent melting points rang- MICs of the most active compounds towards B. anthracis,
ing from 36.2 to 41.6 °C, which may indicate the presence B. cereus, B. subtilis, E. faecalis and L. monocytogenes are
of concomitant polymorphs.[25]
presented in Table 4. The dodecyl α-l-glycoside 20 was the
Table 3. Antibacterial activities expressed as the diameter of the inhibition zones (Ϯstandard deviation) for compounds 7, 9, 14–16 and
18–20 and comparison with the activities of the control (chloramphenicol) using the paper disc diffusion method.[a,b]
Bacteria
Diameter of inhibition [mm]
7[8]
9[8]
14
15
16
18[8]
19[8]
20[8]
Control[c]
I
II
B. cereus
B. subtilis
11Ϯ1
12Ϯ1
14Ϯ0
Ͻ6.4
Ͻ6.4
Ͻ6.4
Ͻ6.4
Ͻ6.4
10Ϯ0
10Ϯ0
17Ϯ2
Ͻ6.4
Ͻ6.4
Ͻ6.4
Ͻ6.4
Ͻ6.4
13Ϯ3
14Ϯ1
11Ϯ1
8Ϯ0
10Ϯ1
10Ϯ1
8Ϯ1
11Ϯ3
13Ϯ1
9Ϯ0
Ͻ6.4
9Ϯ1
9Ϯ1
9Ϯ1
10Ϯ1
58Ϯ16
28Ϯ12
23Ϯ12
Ͻ6.4
17Ϯ7
Ͻ6.4
12Ϯ2
12Ϯ2
10Ϯ0
8Ϯ1
10Ϯ1
8Ϯ0
12Ϯ2
10Ϯ0
9Ϯ2
9Ϯ0
10Ϯ1
9Ϯ0
27Ϯ5
25Ϯ3
13Ϯ2
Ͻ6.4
12Ϯ2
Ͻ6.4
Ͻ6.4
9Ϯ1
25Ϯ3
30Ϯ3
29Ϯ4
29Ϯ3
32Ϯ4
Ͻ6.4
40Ϯ5
46Ϯ3
39Ϯ4
42Ϯ4
45Ϯ3
22Ϯ2
42Ϯ3
42Ϯ6
E. faecalis
E. coli
L. monocytogenes
P. aeruginosa
S. enteritidis
S. aureus
Ͻ6.4
8Ϯ0
Ͻ6.4
11Ϯ1
Ͻ6.4
12Ϯ3
30Ϯ3
27Ϯ5
12Ϯ1
[a] A solution of the compound (300 μg) in DMSO (15 μL) was applied on the disk. [b] The inhibition diameter was Ͻ6.4 mm for all the
bacteria tested for compounds 6,[8] 17 and 21.[8] Compound 8 was also considered inactive (inhibition diameters of 9 and 10 mm for B.
subtilis and L. monocytogenes).[8] [c] Solutions of chloramphenicol in DMSO (15 μL) were used as controls (I: 30 μg; II: 300 μg).
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Tuning the Bioactivity of Deoxy Glycosides to Structure
most effective compound tested, showing a MIC value of ing as scaffolds for future developments because no com-
25 μm with Bacillus spp., followed by its enantiomer 16. The mercial drugs are available that selectively inhibit BChE. To
β-glycosides 7 and 9 were ineffective against the Bacillus assess the type of inhibition of compound 6, the kinetic
species, presenting a MIC of only 112 and 93 μm, respec- parameters KmЈ and VЈ (apparent Km and V, respectively)
tively, with E. faecalis.
were determined for increasing inhibitor concentrations. VЈ
did not show any significant change, whereas the values of
KmЈ increased. These results suggest that this compound is
a competitive inhibitor of BChE and the competitive inhibi-
tor constant Ki = 0.36 mm was calculated from the intercept
of the straight line with the inhibitor axis plotted in Fig-
ure 3. None of the alkyl 2,6-dideoxy glycosides tested, nor
the compounds belonging to the 2-deoxy-β-d series, re-
vealed significant inhibitory activity towards BChE.
Table 4. Antibacterial activities expressed as MIC for compounds
7, 9, 16 and 20 in comparison with the activities of the control
(chloramphenicol).[a]
Bacteria
MIC [mm]
7
9
16
20
Control
0.025
B. anthracis (pathogenic)
B. anthracis (vacinal)
B. anthracis (ovine)
B. cereus
n.d.
n.d.
n.d.
Ͼ0.770 0.050 0.025
Ͼ0.770 0.050 0.025
Ͼ0.770 0.050 0.025
0.025
0.025
Ͼ1.809[8] Ͼ1.504[8] 0.025 0.025[8] 0.019
B. subtilis
E. faecalis
L. monocytogenes
Ͼ1.809[8] Ͼ1.504[8] 0.050 0.025[8] 0.009
0.112[8] 0.093[8] 0.050 0.050[8] 0.019
n.d.
0.752[8] 0.050 0.100[8] 0.019
[a] MICs for the B. anthracis strains were determined by the agar
dilution method (concentrations ranging from 1–256 μg/mL),
whereas the MICs for the remaining bacteria were evaluated by the
broth dilution method (concentrations ranging from 1–500 μg/mL).
The mechanism of action of the synthesized alkyl glycos-
ides is not clear, but preliminary work performed by our
group on B. cereus suggests that these compounds may in-
teract with the cellular membrane, altering its permeability
and causing autolysis followed by cell death.[13] This hy-
pothesis is supported by the surface activity results. The
CMC of dodecyl 2,6-dideoxy-α-d-arabino-hexopyranoside
(16) is the lowest observed (Table 1) whereas dodecyl 2,6-
dideoxy-α-l-arabino-hexopyranoside (20) presents the high-
est surface excess. The latter parameter is indicative of a
higher adsorption tendency at interfaces, which indicates
that slightly higher drug concentrations on the Bacillus
membrane will be found. This minor difference between
these parameters for the two enantiomers, if not lying
within the uncertainties, could account for the higher MIC
obtained for 16 (16 μg/mL, 50 μm) compared with that of
its l enantiomer 20 (8 μg/mL, 25 μm).
Figure 3. Plot of KmЈ/VЈ versus concentration of octyl 2-deoxy-α-
d-glycoside 6 (KmЈ is the apparent Km and VЈ is the apparent V).
The intercept of the resulting straight line with inhibitor axis gives
a value of –Ki.
Molecular Docking Studies
The molecular docking calculations of the most active
glycoside 6, its β anomer 7 and the corresponding 2,6-dide-
oxy compounds 14 and 15 were performed with BChE to
rationalize the selectivity observed for compound 6. Even
though the interaction energies are similar (ca. –7 kcal/
mol), we observed very different modes of binding. Our
cluster analysis generated up to 250 conformational clusters
and we visually analysed the most abundant. These were
grouped by their specific mode of binding to the different
residues in the active site. Indeed, ligand 6 presented a low-
est-energy conformation with several interactions with the
two accessible residues of the catalytic triad, namely Ser-
Anticholinesterase Activity
The AChE and BChE inhibitory activities of some of 198 and His-438 (see Figure 4, a). The high abundance of
the synthesized alkyl glycosides and those of the standards, this mode of binding was confirmed by the cluster analysis
rivastigmine and donepezil, two drugs used to treat Alzhei- of the conformational data obtained (ca. 61% of the solu-
mer’s disease, are now discussed. None of the tested com- tions show a direct interaction with the catalytic residues).
pounds significantly inhibited AChE, but at a concentration
Interestingly, it is the most flexible hydroxy group (bound
of 100 μg/mL, the octyl 2-deoxy-α-d-glycoside 6 caused to C-6) that is placed at a hydrogen-bonding distance to
100% inhibition of BChE and compound 8, its dodecyl α- both catalytic residues, stabilizing the protein–inhibitor
d homologue, caused 81% inhibition at the same concentra- complex. Even though the active site cavity is significantly
tion. The final concentration of the tested compounds was large in BChE, a complex of this type completely blocks
ten-fold higher than that of the non-selective BChE inhibi- the substrate access to the catalytic triad. Ligand 7 is the β
tors rivastigmine and donepezil, which showed 100 and stereoisomer of 6 and its binding mode changes with this
75% inhibition, respectively, of BChE. However, the struc- geometric constraint (Figure 4, b). With this change in an-
tures of these glycoside inhibitors seem to be quite promis- omeric orientation, the ligand moves away from His-438
Eur. J. Org. Chem. 2013, 1448–1459
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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1453

A. P. Rauter et al.
FULL PAPER
these observations are based on rigid crystal structures,
there is significant evidence that the selective inhibition of
BChE by compound 6 is a result of this size-exclusion
mechanism.
Figure 4. Lowest-energy solutions from the docking calculations of
the deoxy glycoside ligands (grey) 6 (a), 7 (b), 14 (c) and 15 (d) in
BChE (green). Interactions are represented by black dashed lines.
The catalytic residues are shown with the carbon atoms represented
in pink.
Figure 5. Structural representation of the amino acids around the
active site cavity that differ in the protein sequences of both BChE
(green)[26] and AChE (grey).[27] The main chains of the proteins are
shown as ribbons. The catalytic residues are shown as spheres with
the carbon atoms represented in pink.
while maintaining a direct interaction with Ser-198. This
interaction remains the most abundant (ca. 54%).
Glycoside 14 differs from 6 as it bears a second deoxy
position at C-6, and this has a significant impact on its
binding mode to BChE (Figure 4, c). Removal of this key
OH group induces a strong slide of the ligand away from
the catalytic triad, which results in the absence of interac-
tions with any of these residues in many conformational
clusters. The β stereoisomer of 14 is ligand 15, which seems
to bind to BChE similarly to the previous β ligand 7. In the
absence of the OH group at the 6-position, this ligand
adopts a conformation that favours the interaction with
Ser-198 through the endocyclic oxygen (Figure 4, d). Unlike
glycoside 14, the interaction of compound 15 with Ser-198
renders a low-energy conformational cluster, which contrib-
utes significantly to its predominant mode of binding (ca.
50%).
These results show significant differences in the modes
of binding of the glycosides studied, with compound 6 pres-
enting the most promising interactions. In fact, this ligand
displays a network of hydrogen bonds with all the accessible
residues of the catalytic triad, which may explain the higher
inhibition determined experimentally. Although compound
6 displayed significant inhibition of BChE, it was not effec-
tive in the assays with AChE. To rationalize these experi-
mental results, similar docking studies of 6 on AChE were
performed. However, the observed binding modes were not
significantly different to account for the contrasting inhibi-
tion profiles obtained. These puzzling results prompted us
to analyse in detail the access to the active site cavities of
BChE and AChE.
Figure 6 shows the lowest-energy solutions of the first 10
clusters obtained in the docking calculations of compound
6 in BChE and AChE. The docking solutions of BChE con-
centrate on the catalytic residues in a cavity that is large
enough for the inhibitor to adopt its preferred conforma-
tion and form stable enzyme–inhibitor complexes. Access
to the AChE active site is partially occluded resulting in a
significant amount of the docking clusters outside the cav-
ity of interest. The difficulty in generating docking solutions
closer to the catalytic triad together with the narrow en-
trance to the active site can generate a kinetic barrier for
these compounds to access their target. Such a barrier will
have an impact on the activity/inhibition profiles of the en-
zyme and may explain the inhibition results obtained for
deoxy glycoside 6 with AChE.
Figure 5 illustrates the differences between the two X-ray
structures.[26,27] The catalytic triad structure is conserved,
but there are major differences in the size and shape of the
active site entrance. AChE exhibits several bulky residues
Figure 6. Lowest-energy solutions for the first 10 clusters obtained
in the docking calculations of compound 6 (cyan) in BChE (left)
and AChE (right). The protein surface is shown in grey to illustrate
the active site cavity. The catalytic residues are shown with carbon
that hinder access to the active site cavity. Even though atoms represented in pink.
1454
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© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2013, 1448–1459

Tuning the Bioactivity of Deoxy Glycosides to Structure
TLC was carried out on aluminium sheets (20 cmϫ20 cm) coated
with 0.2 mm silica gel 60 F-254 (Merck) and detection was ac-
complished by spraying the plates with a solution of H2SO4 in eth-
anol (10%) followed by heating at 120 °C.
Conclusions
Alkyl 2-deoxy- and 2,6-dideoxy-arabino-hexopyranosides
have been explored from a multidisciplinary perspective.
Organic and physical chemistry techniques were used to
produce new molecules and correlate their properties with
their biological behaviour.
The compounds were purified by column chromatography (CC)
using silica gel 60G (0.040–0.063 mm, Merck) or silica gel 60G
(0.015–0.040 mm, Merck)
To estimate the most important physical phenomena that
contribute to the antimicrobial activities exhibited by this
new family of antibiotics, parameters related to molecule
aggregation and adsorption at the air/water interface were
evaluated. It was found that removal of the hydroxy group
at the 6-position is crucial to increasing aggregation over
adsorption. The decrease observed in the CMC/C20 ratio
on going from octyl to dodecyl dideoxy glycosides suggests
a better packing efficiency of the l series glycosides. How-
ever, these results are within experimental uncertainties. The
α-anomeric configuration and the 2,6-dideoxygenation
pattern proved to be essential for antibacterial activity
against the Bacillus species tested. The l configuration en-
hanced this activity, particularly towards Bacillus anthracis.
On the other hand, the presence of the primary hydroxy
group at C-6 was found to be crucial for the inhibition of
BChE by octyl 2-deoxy-α-d-glycoside 6 and its dodecyl α-
d homologue 8. The inhibition type was assessed for the
most active glycoside 6 and it was found to be competitive.
Docking studies have helped us to understand the role of
the flexible hydroxy group in the active site and to rational-
ize the lack of activity of the β anomers as well as that of
the corresponding dideoxy α anomers. The observed selec-
tivity towards BChE was unexpected due to the similarities
between the BChE and AChE active sites. However, it could
be explained by the physical hindrances experienced by the
inhibitor when accessing the active site of AChE.
This work highlights the importance of organic chemis-
try in tuning alkyl glycoside bioactivity through structural
features such as the d or l configuration, the deoxygenation
pattern and the orientation of the anomeric substituent.
Furthermore, the adopted methodology for the synthesis of
the studied deoxy glycosides has the advantage of delivering
the α anomer as the major reaction product, which show
the greatest bioactivity. In addition, new leads have been
reported that encourage further study of these molecular
entities aiming at the development of new drug candidates
with new mechanisms of action to overcome antibiotic re-
sistance and/or neurodegenerative impairments, two major
concerns in today’s society.
Melting points were first obtained with an SMP3 Melting Point
Apparatus, Stuart Scientific, Bibby. Elemental analyses were per-
formed by the Microanalysis Service of the Instituto Superior
Técnico, Universidade Técnica de Lisboa. Optical rotations were
measured with a Perkin–Elmer 343 polarimeter.
NMR spectra were recorded with a Bruker Avance 400 spectrome-
ter at 298 K operating at 100.62 MHz for 13C NMR and at
400.13 MHz for 1H NMR. The solvents used were CDCl3 with
0.03% TMS and CD3OD (Sigma–Aldrich). The chemical shifts are
reported as δ (ppm) and the coupling constants (J) are given in Hz.
General Procedure for the Tosylation Reaction: TsCl (3.3 mmol) and
pyridine (3 mL) were added to a solution of the alkyl 2-deoxy-d-
arabino-hexopyranoside (1.5 mmol) in CH2Cl2 (3 mL) at 0 °C and
the reaction mixture was stirred at room temp. for 2 h. The solution
was washed with a satd. solution of NaHCO3 and the organic
phase was washed with water and dried with sodium sulfate. After
filtration, the organic phase was evaporated in the rotavapor to give
a residue that was purified by CC eluting with EtOAc/cyclohexane
(1:1) to afford the product.
Octyl 2-Deoxy-6-O-tosyl-α-D-arabino-hexopyranoside (10): The
above-mentioned procedure gave 10 as a syrup (0.57 g, 88.0%).
α2D0 = +2.2 (c = 1, CH2Cl2). Rf = 0.49 (1:1 EtOAc/cyclohexane). 1H
NMR (400 MHz, CDCl3): δ = 7.80 (d, 2 H, Ph-Ts), 7.34 (d, 2 H,
3
2
Ph-Ts), 4.80 (d, J1,2a = 2.78 Hz, 1 H, 1-H), 4.36 (dd, J6a,6b
=
3
3
2
10.86, J6a,5 = 4.55 Hz, 1 H, 6a-H), 4.24 (dd, J6b,5 = 1.01, J6a,6b
= 10.86 Hz, 1 H, 6b-H), 3.92 (ddd, J3,2a = 9.6, J3,2e = 5.00, J4,3
= 9.1 Hz, 1 H, 3-H), 3.69 (ddd, J5,6b = 1.01, J5,6a = 4.30, J4,5
3
3
3
3
3
3
=
=
2
3
9.1 Hz, 1 H, 5-H), 3.51 (ddd, J1Јa,1Јb = 9.6, J1Јa,2Јa
=
3J1Јa,2Јb
6.95 Hz, 1 H, 1Јa-H), 3.38 (t, 3J4,3 = 3J4,5 = 9.1 Hz, 1 H, 4-H), 3.23
(ddd, 3J1Јb,2Јb = 3J1Јb,2Јa = 6.82, 2J1Јa,1Јb = 9.6 Hz, 1 H, 1Јb-H), 2.43
2
3
(s, 3 H CH3-Ts), 2.08 (dd, J2e,2a = 12.88, J2e,3 = 4.8 Hz, 1 H, 2e-
2
2
H), 1.62 (td, J2a,3 = 9.6, J2e,2a = 12.88 Hz, 1 H, 2a-H), 1.52–1.48
(m, 2-H, 2Јa-H, 2Јb-H), 1.31–1.23 (m, 10 H, 3Јa,b-H to 7Јa,b-H),
0.88 (t, 3J7Ј,8Ј = 6.82 Hz, 3 H, 8Ј-H) ppm. 13C NMR (100.62 MHz,
CDCl3): δ = 144.5, 132.7, 129.8, 127.9 (Ph-Ts), 101.9 (C-1), 78.8
(C-4), 73.9 (C-5), 73.3 (C-3), 71.1 (C-1Ј), 63.7 (C-6), 41.2 (C-2),
33.8, 31.5, 31.3, 31.2, 27.9, 24.5 (C-2Ј to C-7Ј), 15.2 (C-8Ј) ppm.
C21H34O7S (430.20): calcd. C 58.58, H 7.97; found C 58.55, H 7.95.
Octyl 2-Deoxy-6-O-tosyl-β-D-arabino-hexopyranoside (11): The
above-mentioned procedure gave 11 as a syrup (0.54 g, 84.3%).
α2D0 = –5.1 (c = 1, CH2Cl2). Rf = 0.45 (1:1 EtOAc/cyclohexane). 1H
NMR (400 MHz, CDCl3): δ = 7.80 (d, 2 H, Ph-Ts), 7.34 (d, 2 H,
3
2
Ph-Ts), 4.47 (d, J1,2a = 8.08 Hz, 1 H, 1-H), 4.36 (dd, J6a,6b
=
3
3
2
10.86, J6a,5 = 4.55 Hz, 1 H, 6a-H), 4.24 (dd, J6b,5 = 1.01, J6a,6b
= 10.86 Hz,1 H, 6b-H), 3.76 (ddd, 3J1Јa,2Јb = 3J1Јa,2Јa = 6.69, 2J1Јa,1Јb
3
3
3
= 9.35 Hz, 1 H, 1Јa-H), 3.64 (ddd, J3,4 = 9.1, J3,2a = 9.6, J3,2e
=
Experimental Section
4.8 Hz, 1 H, 3-H), 3.40–3.34 (m, 3 H, 4-H, 5-H, 1Јb-H), 2.43 (s, 3
2
3
General: Starting materials and reagents were purchased from
Sigma–Aldrich, Fluka and Acros. The solvents were dried prior to
use with 4 or 3 Å (methanol) molecular sieves. Compounds 2–9
were synthesized according to the procedures previously described
by us.[8] The spectroscopic and physical data of these compounds
are in agreement with those previously reported, with the exception
H, CH3-Ts), 2.08 (dd, J2e,2a = 12.88, J2e,3 = 4.8 Hz, 1 H, 2e-H),
3
2
1.62 (td, J2a,3 = 9.6, J2e,2a = 12.88 Hz, 1 H, 2a-H), 1.52–1.48 (m,
2 H, 2Јb-H), 1.31–1.23 (m, 10 H, 3Јa,b-H to 7Јa,b-H), 0.88 (t, 3J7Ј,8Ј
= 6.82 Hz, 3 H, 8Ј-H) ppm. 13C NMR (100.62 MHz, CDCl3): δ =
144.5, 132.7, 129.8, 127.9 (Ph-Ts), 101.9 (C-1), 78.8 (C-5), 73.9 (C-
4), 73.3 (C-3), 69.6 (C-1Ј), 69.0 (C-6), 41.2 (C-2), 33.8, 31.5, 31.3,
31.2, 27.9, 24.5 (C-2Ј to C-7Ј), 15.2 (C-8Ј) ppm. C21H34O7S
(430.20): calcd. C 58.58, H 7.97; found C 58.57, H 7.93.
of the specific rotation of compound 7, which is corrected to α2D0
–1.3 (c = 1, CH2Cl2).
=
Eur. J. Org. Chem. 2013, 1448–1459
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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1455

A. P. Rauter et al.
FULL PAPER
Dodecyl 2-Deoxy-6-O-tosyl-α-
D
-arabino-hexopyranoside (12): The
80 °C. α2D0 = –4.4 (c = 1, CH2Cl2). Rf = 0.45 (3:1 EtOAc/cyclohex-
3
above-mentioned procedure gave 12 as a syrup (0.68 g, 92.9%).
α2D0 = +2.4 (c = 1, CH2Cl2). Rf = 0.53 (1:1 EtOAc/cyclohexane). 1H
NMR (400 MHz, CDCl3): δ = 7.82 (d, 2 H, Ph-Ts), 7.33 (d, 2 H,
ane). 1H NMR (400 MHz, CDCl3): δ = 4.47 (d, J1,2a = 8.08 Hz, 1
3
3
3
H, 1-H), 3.86 (ddd, J3,4 = 8.34, J3,2e = 2.53, J3,2a = 9.35 Hz, 1
2
H, 3-H), 3.65–3.56 (m, 2 H, 5-H, 1Јa-H), 3.43 (dt, J1Јb,1Јa = 9.35,
3
2
3
Ph-Ts), 4.81 (d, J1,2a = 2.27 Hz, 1 H, 1-H), 4.40 (dd, J6a,6b
=
3J1Јb,2Јa
=
3J1Јb,2Јb = 7.07 Hz, 1 H, 1Јb-H), 3.10 (t, J4,3
=
3J4,5
=
3
3
2
3
2
10.86, J6a,5 = 4.30 Hz, 1 H, 6a-H), 4.21 (dd, J6b,5 = 1.01, J6a,6b
8.34 Hz, 1 H, 4-H), 2.10 (dd, J3,2e = 2.53, J2e,2a = 12.44 Hz, 1 H,
1 H, 2e-H), 1.65–1.56 (m, 3 H, 2a-H, 2Јa-H, 2Јb-H), 1.35–1.23 (m,
13 H, 3Јa,b-H to 7Јa,b-H, 6-CH3), 0.88 (t, J7Ј,8Ј = 5.81 Hz, 3 H,
3
3
3
= 10.86 Hz, 1 H, 6b-H), 3.95 (ddd, J3,2a = 9.6, J3,2e = 5.05, J4,3
= 9.1 Hz, 1 H, 3-H), 3.69 (ddd, J5,6b = 1.01, J5,6a = 4.30, J4,5
3
3
3
3
=
=
2
3
9.1 Hz, 1 H, 5-H), 3.52 (ddd, J1Јa,1Јb = 9.6, J1Јa,2Јa
=
3J1Јa,2Јb
8Ј-H) ppm. 13C NMR (100.62 MHz, CDCl3): δ = 99.5 (C-1), 77.6
(C-5), 71.8 (C-4), 71.5 (C-3), 39.1 (C-1Ј), 31.8 (C-2), 29.7, 29.6,
29.4, 29.2, 26.0, 22.6 (C-2Ј to C-7Ј), 17.7 (C-6), 14.1 (C-8Ј) ppm.
C14H28O4 (260.20): calcd. C 64.58, H 10.84; found C 64.60, H
10.83.
6.95 Hz, 1 H, 1Јa-H), 3.42 (t, 3J4,3 = 3J4,5 = 9.1 Hz, 1 H, 4-H), 3.28
(ddd, 3J1Јb,2Јb = 3J1Јb,2Јa = 6.82, 2J1Јa,1Јb = 9.6 Hz, 1 H, 1Јb-H), 2.44
2
3
(s, 3 H, CH3-Ts), 2.10 (dd, J2e,2a = 12.88, J2e,3 = 5.05 Hz, 1 H,
3
2
2e-H), 1.64 (td, J2a,3 = 9.6, J2e,2a = 12.88 Hz, 1 H, 2a-H), 1.51–
1.48 (m, 2 H, 2Јa-H, 2Јb-H), 1.32–1.22 (m, 18 H, 3Јa,b-H to 11Јa,b-
Dodecyl 2,6-Dideoxy-α-D-arabino-hexopyranoside (16): The above-
3
H), 0.88 (t, J11Ј,12Ј = 6.32 Hz, 3 H, 12Ј-H) ppm. 13C NMR
mentioned procedure gave 16 as a white solid (0.73 g, 79%). M.p.
(100.62 MHz, CDCl3): δ = 144.5, 132.7, 129.8, 127.9 (Ph-Ts), 97.3
(C-1), 71.5 (C-4), 68.5 (C-6), 69.4 (C-5), 68.5 (C-3), 67.3 (C-1Ј),
37.0 (C-2), 21.6 (CH3-Ts), 31.9, 29.6, 29.6, 29.5, 29.4, 29.3, 26.1
(C-2Ј to C-11Ј), 14.1 (C-12Ј) ppm. C25H42O7S (486.27): calcd. C
61.70, H 8.70; found C 61.72, H 8.67.
36 °C. α2D0 = +7.1 (c = 1, CH2Cl2). Rf = 0.53 (3:1 EtOAc/cyclohex-
3
ane). 1H NMR (400 MHz, CDCl3): δ = 4.81 (d, J1,2a = 2.78 Hz, 1
3
3
3
H, 1-H), 3.88 (ddd, J3,2e = 5.05, J3,2a = 11.87, J3,4 = 10.36 Hz,
3
1 H, 3-H), 3.65–3.59 (m, 2 H, 5-H, 1Јa-H), 3.34 (dt, J1Јb,2Јb
=
3J1Јb,2Јa = 6.57, J1Јa,1Јb = 9.10 Hz, 1 H, 1Јb-H), 3.08–3.03 (m, 2 H,
3
2
3
Dodecyl 2-Deoxy-6-O-tosyl-β-
D
-arabino-hexopyranoside (13): The
4-H, 5-H), 2.10 (dd, J2e,2a = 12.76, J2e,3 = 5.05 Hz, 1 H, 2e-H),
3
above-mentioned procedure gave 13 as a syrup (0.66 g, 90.8%).
α2D0 = –5.1 (c = 1, CH2Cl2). Rf = 0.50 (1:1 EtOAc/cyclohexane). 1H
NMR (400 MHz, CDCl3): δ = 7.82 (d, 2 H, Ph-Ts), 7.33 (d, 2 H,
1.66 (td, J2a,3 = 11.87 Hz, 1 H, 2a-H), 1.59–1.52 (m, 2 H, 2Јa-H,
2Јb-H), 1.39–1.20 (m, 21 H, 3Јa,b-H to 11Јa,b-H, 6-CH3), 0.88 (t,
3J7Ј,11Ј = 7.33 Hz, 3 H, 12Ј-H) ppm. 13C NMR (100.62 MHz,
Ph-Ts), 4.49 (dd, 3J1,2a = 9.6, 3J1,2e = 2.27 Hz, 1 H, 1-H), 4.35–4.25 CDCl3): δ = 97.1 (C-1), 77.8 (C-4), 69.0 (C-5), 67.5 (C-3), 67.4 (C-
3
3
3
(m, 2 H, 6a-H, 6b-H), 3.69 (ddd, J1Јa,2Јa = J1Јa,2Јb = 6.19, J1Јa,1Јb
1Ј), 37.8 (C-2), 31.8, 29.6, 29.6, 29.5, 29.4, 29.3, 26.1, 22.7 (C-2Ј to
= 9.22 Hz, 1 H, 1Јa-H), 3.56 (m, 1 H, 3-H), 3.43–3.28 (m, 3 H, 4- C-11Ј), 17.7 (C-6), 14.1 (C-12Ј) ppm. C18H36O4 (316.26): calcd. C
2
H, 5-H, 1Јb-H), 2.44 (s, 3 H, CH3-Ts), 2.10 (dd, J2e,2a = 12.88,
68.31, H 11.47; found C 68.32, H 11.46.
Dodecyl 2,6-Dideoxy-β- -arabino-hexopyranoside (17): The above-
mentioned procedure gave 17 as a white solid (0.69 g, 75.0%). M.p.
97 °C. α2D0 = –4.5 (c = 1, CH2Cl2). Rf = 0.50 (3:1 EtOAc/cyclohex-
ane). 1H NMR (400 MHz, CDCl3): δ = 4.49 (dd, 3J1,2a = 9.6, 3J1,2e
3J2e,3 = 5.05 Hz, 1 H, 2e-H), 1.57–1.48 (m, 3 H, 2a-H, 2Јa-H, 2Јb-
H), 1.32–1.22 (m, 18 H, 3Јa,b-H to 11Јa,b-H), 0.88 (t, J11Ј,12Ј
D
3
=
6.32 Hz, 3 H, 12Ј-H) ppm. 13C NMR (100.62 MHz, CDCl3): δ =
144.5, 132.7, 129.8, 127.9 (Ph-Ts), 99.3 (C-1), 69.8 (C-1Јa), 69.0 (C-
6), 71.1 (C-3), 71.3, 73.5 (C-4, C-5), 37.2 (C-2), 21.6 (CH3-Ts), 31.9,
29.6, 29.5, 29.4, 29.3, 26.1 (C-2Ј to C-11Ј), 14.1 (C-12Ј) ppm.
C25H42O7S (486.27): calcd. C 61.70, H 8.70; found C 61.72, H 8.67.
3
3
3
= 1.26 Hz, 1 H, 1-H), 3.86 (dt, J1Јa,1Јb = 9.22, J1Јa,2Јa = J1Јa,2Јb
=
=
=
3
3
3
6.95 Hz, 1 H, 1Јa-H), 3.59 (ddd, J3,4 = 9.09, J3,2e = 2.02, J3,2a
9.35 Hz, 1 H, 3-H), 3.43 (dt, J1Јb,1Јa = 9.22, J1Јb,2Јa = J1Јb,2Јa
7.07 Hz, 1 H, 1Јb-H), 3.26 (dq, J5,6 = 6.44, J5,4 = 9.09 Hz, 1 H,
5-H), 3.09 (t, J5,4 = 9.09 Hz, 1 H, 4-H), 2.21 (ddd, J1,2e = 1.52,
3
3
3
3
3
General Procedure for the Deoxygenation Reaction: LiAlH4
3
3
(10.2 mmol, 0.39 g) was added to a solution of the alkyl 2-deoxy-
6-O-tosyl-d-arabino-hexopyranoside (2.9 mmol) in THF (44 mL) at 3J3,2e = 2.02, 2J2e,2a = 12.88 Hz, 1 H, 2e-H), 1.62–1.55 (m, 3 H, 2a-
0 °C and the reaction mixture was stirred at room temp. for 72 h.
Then EtOAc was added dropwise until no gas was formed, then
ice flakes were added to dissolve the precipitate and finally CH2Cl2
(50 mL) was added. The organic phase was washed with water,
dried with sodium sulfate and evaporated in a rotavapor to give a
residue that was purified by CC eluting with EtOAc/cyclohexane
(5:1) to afford the final product.
H, 2Јa-H, 2Јb-H), 1.35–1.24 (m, 21 H, 3Јa,b-H to 11Јa,b-H, 6-
3
CH3), 0.88 (t, J11Ј,12Ј = 6.82 Hz, 3 H, 12Ј-H) ppm. 13C NMR
(100.62 MHz, CDCl3): δ = 99.5 (C-1), 77.6 (C-5), 71.7 (C-4), 71.6
(C-3), 69.6 (C-1Ј), 39.1 (C-2), 31.9, 30.9, 29.6, 29.6, 29.5, 29.4, 29.3,
26.0 (C-2Ј to C-11Ј), 17.7 (C-6), 14.1 (C-12Ј) ppm. C18H36O4
(316.26): calcd. C 68.31, H 11.47; found C 64.29, H 10.48.
Surface Activity: The surface tensions were determined with a
Kruss K100MK2 automatic Interfacial Tensiometer using the
du Noüy ring method at 35.0Ϯ0.1 °C. The du Noüy ring non-de-
tachment technique was used and the surface tensions determined
as a function of surfactant concentration following the experimen-
tal procedure described previously. The Harkins and Jordan correc-
Octyl 2,6-Dideoxy-α-D-arabino-hexopyranoside (14): The above-
mentioned procedure gave 14 as a syrup (0.53 g, 70.0%). α2D0 = +3.5
(c = 1, CH2Cl2). Rf = 0.49 (3:1 EtOAc/cyclohexane). 1H NMR
3
(400 MHz, CDCl3): δ = 4.81 (d, J1,2a = 3.03 Hz, 1 H, 1-H), 3.87
3
3
(ddd, J3,4 = 9.35, J3,2a = 12.25, 3J2e,3 = 5.30 Hz, 1 H, 3-H), 3.64–
3
3.57 (m, 2 H, 5-H, 1Јa-H), 3.34 (dt, J1Јb,2Јb
=
3J1Јb,2Јa = 6.82, tion was automatically introduced by the tensiometer.
2J1Јa,1Јb = 9.35 Hz, 1 H, 1Јb-H), 3.05 (t, J4,3 = J4,5 = 9.35 Hz, 1
3
3
Most aqueous solutions were prepared by weight in an A&D In-
struments analytical balance to Ϯ2ϫ10–5 g and the dissolution of
the most concentrated solutions was accelerated by immersing the
solutions in an ultrasound bath at 35 °C. The surface tensions were
determined exclusively on clear isotropic solutions and the tensi-
ometer calibration was checked with Milli-Q water before each run.
The most dilute solutions exhibited a time-dependent surface ten-
sion that decreases on standing. Therefore all solutions were pre-
equilibrated for at least 30 min prior to measurement. The surface
tension values given in this work are the average of at least 10
independent evaluations and include an evaluation of solutions
H, 4-H), 2.10 (dd, 2J2e,2a = 12.63, 3J2e,3 = 5.30 Hz, 1 H, 2e-H), 1.66
3
2
3
(td, J2a,3 = 12.25, J2e,2a = 12.63, J1,2a = 3.03 Hz, 1 H, 2a-H),
1.60–1.53 (m, 2 H, 2Јa-H, 2Јb-H), 1.39–1.20 (m, 10 H, 3Јa,b-H to
7Јa,b-H, 6-CH3), 0.88 (t, J7Ј,8Ј = 6.82 Hz, 3 H, 8Ј-H) ppm. 13C
3
NMR (100.62 MHz, CDCl3): δ = 97.1 (C-1), 77.7 (C-4), 68.9 (C-
5), 67.5 (C-3), 67.4 (C-1Ј), 37.8 (C-2), 31.8, 29.4, 29.3, 29.2, 26.1,
22.6 (C-2Ј to C-7Ј), 17.7 (C-6), 14.0 (C-8Ј) ppm. C14H28O4 (260.20):
calcd. C 64.58, H 10.84; found C 64.56, H 10.85.
Octyl 2,6-Dideoxy-β-
mentioned procedure gave 15 as a white solid (0.54 g, 72.0%). M.p.
D
-arabino-hexopyranoside (15):[28] The above-
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Tuning the Bioactivity of Deoxy Glycosides to Structure
prepared independently by weight and by successive dilution to
cross-check the reliability of the experimental procedure.
serum; substrates: acetylthiocholine iodide (ATchI) and S-butyryl-
thiocholine iodide (BTchI) crystalline, and 5,5Ј-dithiobis(2-nitro-
benzoic acid) (DTNB); reagents used for preparing buffers and
solutions: KH2PO4, KOH, NaHCO3. Deionized/sterilized water
was used to prepare the pH 8.0 buffer, DTNB and test compound
solutions.
Antimicrobial Activity: The antibacterial activities of the com-
pounds were screened by using the paper disk diffusion method.[8]
The tests were carried out with Bacillus cereus (ATCC 11778), Ba-
cillus subtilis (ATCC 6633), Enterococcus faecalis (ATCC 29212),
Solution Preparations: Preparation of 0.1 m phosphate buffer
(pH 8.0): Potassium dihydrogen phosphate (136.1 mg, 1 mmol) was
dissolved in water (10 mL) and adjusted with potassium hydroxide
to a pH of 8.0Ϯ0.1. The buffer solution was freshly prepared and
stored in the refrigerator.
Escherichia
(ATCC 7644), Pseudomonas aeruginosa (ATCC 27853), Salmonella
enteritidis (ATCC 13076) and Staphylococcus aureus
coli
(ATCC 8739),
Listeria
monocytogenes
(ATCC 25923). The culture medium and used for bacteria growth
was Nutrient Agar incubated at 37 °C. Paper disks of 6.4 mm were
placed on the agar and a solution of each substance (300 μg) in
DMSO (15 μL) was applied to each disk. Chloramphenicol was
used as a control. After incubation, the nearest diameter of the
inhibition zone was measured and a compound was considered
active at an inhibition diameter Ն11 mm. At least three replicates
were made.
AChE solution (1.32 U/mL): The enzyme (1.32 U) was diluted in
freshly prepared pH 8.0 buffer until a final volume of 1 mL.
BChE solution (0.44 U/mL): The enzyme (0.44 U) was diluted in
freshly prepared pH 8.0 buffer until a final volume of 1 mL.
DTNB solution (0.01 m): DTNB (3.96 mg, 0.01 mmol) was dis-
solved in water (1 mL) containing sodium hydrogen carbonate
(1.5 mg).
Subsequent tests to determine the minimal inhibition concentra-
tions (MICs) were conducted on the compounds that exhibited
considerable activity by the paper disk diffusion method. The broth
dilution method[8] was used to test the susceptible microbes B. sub-
tilis, B. cereus, E. faecalis and L. monocytogenes. Overnight cultures
were also used. Serial dilutions starting at 500 μg/mL until 1.95 μg/
mL were made for the compounds tested. Chloramphenicol di-
lutions ranged between 50 and 0.195 μg/mL. Bacteria were incu-
bated at 35 °C for 16–20 h. At least three replicates were made.
ATchI and BTchI aqueous solutions (0.022 m) were prepared. All
solutions were stored in Eppendorf caps (100 μL aliquots) in the
refrigerator. Test compounds were prepared with a final concentra-
tion of 100 μg/mL in the reaction cuvette.
AChE/BChE Activity Assay: The buffer solution (200 μL) was
added to the cuvette, followed by the enzyme solution (5 μL),
DTNB (5 μL) and the test compound solution (5 μL). The reagents
were carefully mixed and kept at 30 °C for 15 min in a heated water
bath and then the substrate (5 μL) was added. The absorbance data
was recorded during a reaction time of 4 min at 30 °C. At least
four replicates were made. Blank assays without the enzyme or test
compounds were performed to check for any non-enzymatic hy-
drolysis of the substrate.
The microbial susceptibility of Bacillus anthracis to the most active
compounds was evaluated. Three strains from the collection of the
Instituto Nacional de Saúde Doutor Ricardo Jorge, INSA, Portu-
gal, were used in the tests: BA01, from sheep origin; BA02, human
pathogen; BA03, vaccine strain. The assays were carried out in a
biosafety level 3 laboratory by using the agar dilution method on
the Müller–Hinton medium according to the Clinical and Labora-
tory Standards Institute (CLSI) guidelines.[29]
The final concentrations of chemicals in the test were as follows:
[AChE] = 0.03 U/mL, [BChE] = 0.01U/mL, [Compound] = 100 μg/
mL, [DTNB] = 0.0002273 m, [ATchI] = [BTchI] = 0.0005 m.
Serial dilutions from 256 to 1 μg/mL for each compound and the
standard antibiotic chloramphenicol were prepared in DMSO. The
plates were incubated overnight at 37 °C for 16 h. The MIC is de-
fined as the lowest concentration of the tested compounds that
inhibits bacterial growth.
Statistical Data Analysis: The t-test (one sided) was carried out to
evaluate whether the average inhibition of the enzymes by the new
compounds (100 μg/mL) and the positive control donepezil (ap-
proved drug) are significantly higher than the average inhibition
(0%) obtained in the assay without any inhibitor. The t-test gives
a probability between 0.000 and 1.000. When the probability is
Յ0.050 or 5% then the inhibition obtained with the extract at a
certain concentration is significant.
Anticholinesterase Activity: The colorimetric Ellman’s assay[30] was
used to screen the in vitro anticholinesterase activities of com-
pounds with a concentration of 100 μg/mL. The enzyme activity
and enzyme inhibition were calculated from the change in the rate
of absorbance with time (V = ΔA/Δt) data as follows:
Determination of Inhibition Type and Ki: To determine the inhibi-
tion type and inhibition constant (Ki), the substrate concentration
was varied between 0.03 and 0.13 mm for inhibitor concentrations
between 0 and 1.65 mm. Ki was determined from the plot of KmЈ/
VЈ versus the inhibitor concentration, for which KmЈ is the apparent
Km and VЈ is the apparent V. The intercept of the resulting straight
line with the inhibitor axis is –Ki.
Enzyme inhibition [%] = 100 – enzyme activity [%]
Enzyme activity [%] = 100ϫV/Vmax
Maximum rates (Vmax) were obtained when no inhibitor was used.
V is the rate obtained in the presence of the inhibitor compound.
Spectrophotometer and Chemicals: A double-beam Shimadzu®
spectrophotometer equipped with thermostatic cell holders was
used in the visible range (425 nm) and operated in kinetic mode.
The absorbance data were acquired with a personal computer using
UV Probe software. Appropriate disposable plastic cuvettes were
used in the kinetic experiments. For inhibition type assays, an auto-
mated 96-well microplate reader spectrophotometer (Perlong
DNM-9602) with temperature control at 30 °C was used and mea-
surements were made every minute for 4 minutes.
Computational Methods: Coordinates for the proteins were re-
trieved from the Protein Data Bank (butyrylcholinesterase, BChE:
1P0I;[26] acetylcholinesterase, AChE: 1B41[27]). The files were clean
from everything except the protein (water, ligands and other small
molecules were all removed). There were some missing residues, but
these were located far away from the active site pocket and were
not corrected. The artificial terminals hence generated in the poly-
peptide chain were, for simplicity, treated as neutral terminals. The
modelled deoxy glycoside ligands were built by using PyMOL.[31]
The reagents (p.a. quality) were purchased from Sigma–Aldrich: Each molecule was geometrically optimized with Gaussian 03[32] by
enzymes: AChE from bovine erythrocytes and BChE from human
using the B3LYP functional and 6-31G(d,p) basis sets.[33,34] The
Eur. J. Org. Chem. 2013, 1448–1459
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1457

A. P. Rauter et al.
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Acknowledgments
The authors thank the Management Authorities of the European
Regional Development Fund and the National Strategic Reference
Framework for the support of the Incentive System – Research &
Technological Development Co-Promotion Project nr. 21457. The
Fundação para a Ciência e a Tecnologia (FCT) is gratefully ac-
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