Synthesis and antibacterial activity of ricinoleic acid glycosides

Article abstract of DOI:10.1039/c5ra20136e

The antibacterial properties of twenty-eight novel ricinoleic acid glycosides synthesized by Koenigs-Knorr glycosylation are reported. Seven of them were found to show promising wide spectrum antibacterial activity against Gram positive bacteria of which two compounds, the mannopyranosyl- and the arabinofuranosyl derivatives, were proven effective against various non-clinical/clinical/NorA-overexpressed/resistant strains of Staphylococcus aureus as well as other Gram +ve bacteria such as Bacillus subtilis ATCC 6051 and Micrococcus luteus MTCC 2470. It was found that both the presence of the sugar and its structure are necessary and important for the compounds to be bioactive. The methyl ester protection of the carboxylic acid moiety of the ricinoleic acid unit was also found to be important for imparting good bioactivity to the molecule. Based on the membrane permeability and cell disintegration studies, these compounds are found to increase the bacterial cell membrane permeability, subsequently causing cell death.

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ARTICLE
Synthesis and antibacterial activity of ricinoleic acid glycosides
Ramakrishna Kuppala,a Mugunthan Govindarajan,a Rushikesh Tambat,b Neeraj Patel,c Hemraj
Nandanwar,b,** Kamlesh K. Bhutani,c K. P. Ravindranathan Karthaa,*
Received 00th January 20xx,
Accepted 00th January 20xx
The antibacterial properties of twenty-eight novel ricinoleic acid glycosides synthesized by Koenigs-Knorr glycosylation are
reported. Seven of them were found to show promising wide spectrum antibacterial activity against Gram positive
bacteria of which two compounds, the mannopyranosyl- and the arabinofuranosyl derivatives, were proven effective
against various non-clinical/clinical/NorA-overexpressed/resistant strains of Staphylococcus aureus as well as other Gram
+ve bacteria such as Bacillus subtilis ATCC 6051 and Micrococcus luteus MTCC 2470. It was found that both the presence of
the sugar and its structure are necessary and important for the compounds to be bioactive. The methyl ester protection of
the carboxylic acid moiety of the ricinoleic acid unit was also found important for imparting good bioactivity to the
molecule. Based on the membrane permeability and cell disintegration studies, these compounds are found to be
increasing the bacterial cell membrane permeability and subsequently causing the cell death.
DOI:
10.1039/x0xx00000xwww.rsc.org/
also been demonstrated.15,16,17 Potassium and zinc ricinoleates
Introduction
(2band 4, Fig 1) were also shown to be antibacterial in nature.15
Traditionally, man has relied heavily on natural products for
alleviating their problems associated with diseases and infections
caused by various microbes. It is therefore not surprising that many
of the antibiotics currently in use are natural products or natural
Presently ricinoleic acid and its various salts/derivatives have
reportedly been used in more than 750 commercial products18 in
which they also fulfill their role as emulsion stabilizers, cosmetics,
skin-conditioning agents, etc. Also, mono- and diglycerides of
ricinoleic acid have been reported as potential antimycobacterial
agents.19D’Ocaet al. reported different fatty acid amides and their
structure activity relationship in the context of anti-TB activity and
ricinoleic acid amide (5, Fig 1) was among those exhibited promising
anti-TB activity against M. tb H37Rv.20 More recently, during the
progress of this work, certain Schiff base analogues of methyl
ricinoleate were shown to be good antimicrobials against S.
aureus.21
products-based/inspired. Although, following
a surge in the
development and marketing of antibiotics that was witnessed
during the first three quarters of last century, it was suggested
during the fourth quarter of the century that there was perhaps no
unmet needs in the antibiotic therapeutics,1 a renewed search for
new antibiotic molecules returned by the turn of the century,
largely due to the increasing emergence of instances of drug
resistance.2,3,4In fact antibiotic-resistant infections has now become
a major health concern globally. Thus, the incidents of drug
resistant cases of methicillin resistant Staphylococcus aureus
(MRSA),5,6as well as multidrug drug resistant (MDR) and extensively
drug resistant (XDR) tuberculosis (TB)7 are being frequented
worldwide and hence the demand for identifying drug candidates
with novel modes of action has been growing.7,8Many of the drugs
used for the treatment of bacteria including mycobacterial infection
are cell wall biosynthesis inhibitors.9 The bacterial cell wall
possesses a complex architecture made up of, besides others,
glycans constituted of sugars such as N-acetylglucosamine,
mannose, galactopyranose (Galp), galactofuranose (Galf),
arabinofuranose, ribose and rhamnose,10 and use different
biosynthetic pathways, many of which are validated drug targets,
for the construction of these glycan structures.
OH
OH
Ar
N
O
OR
N
OCH3
O
O
O
1 R = H
2a R = Na
3 R = Li
4 R = 1/2Zn
5
6
2b
R = K
Figure 1: Ricinoleic acid-derived compounds known in the literature
as anti-bacterial and antimycobacterial agents.
The present work was aimed at synthesizing a library of various
glycosides of ricinoleic acid with a view to evaluating their potential
as antibacterial agents. D-Glucose, D-galactopyranose, D-mannose,
N-acetyl-D-glucosamine,
L-rhamnose,
D-arabinose,
D-
galactofuranose and D-lactose were chosen as the sugar substrates
to be linked glycosidically to methyl ricinoleate.
The antimicrobial activity of unsaturated fatty acids such as
oleic/linoleic/linolenic/arachidonic acid, etc, as for example evident
from their use as effective antimicrobial food additives, is well
known.11,12Likewise, ricinoleic acid (12-hydroxy-9-cis-octadecenoic
acid, 1, Fig 1), found in castor oil is also well-known for its medicinal
values and commercial importance. The first report of ricinoleic acid
derivative, for example, sodium ricinoleate (2a, Fig 1) was as a
surface tension depressant on the growth of bacterial culture
media.13,14 Its ability to act as an antibacterial agent against
organisms such as Corynebacterium diphtheria, Gonococci,etc has
Results and discussion
Methyl ricinoleate (7), prepared by reacting 1 with MeOH in the
presence of anhydrous HCl,22 was chosen as the acceptor alcohol
for glycosylation with various acetohalosugars (8) essentially under
the traditional Ag2CO3-AgClO4-mediated Koenigs-Knorr conditions
to afford the desired 1,2-trans-linked glycosides (9) in good yields
(Scheme 1). The structure of these compounds were confirmed by
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NMR spectroscopy in which the J1,2value of 7.9-8.0 Hz was typical, When the free acid was required, the salts obtained were deionized
for example, of the 1,2-trans-linked H-1 and H-2 for the galacto- using Amberlite IR 120 H+ resin (Scheme 1). The compounds
and gluco-configured pyranosides. Likewise, the manno-, arabino-, synthesized (21-48) are shown in Table 1.
rhamno- and the galactofurano-compounds also gave the J1,2 values
characteristic of them (1.5-1.7 Hz, see electronic SI for details). It While acetobromosugars (13-15 and 20) were used as starting
was observed that among the three hexose-derived materials for the synthesis of the hexopyranose-based 21, 24, 28
acetobromosugars (13-15) the comparatively less reactive gluco- and 44 (mono- and disaccharide derivatives), the acetochlorosugar
compound (14), not surprisingly though, did also yield some donors 16 and 19 were found more convenient for the preparation
orthoester (9a) as a by-product (see electronic SI for details). The of the corresponding glucosaminide and the galactofuranoside
acyl group deprotection of these glycoside-derivatives was done by derivatives 32 and 41 as shown in Table 1. On the other hand for
trans-esterification using methanolic NaOMe to afford 10. the rhamnopyranosyl and the arabinofuranosyl ricinoleates35 and
Compound 10 upon aqueous alkaline hydrolysis yielded the desired 38, the easily accessible benzobromo- and benzochlorosugar donors
glycosylated ricinoleic acids 11 in the form of their metal salts 17 and 18, respectively, were utilized for the crucial glycosylation
corresponding to the alkali employed for the hydrolysis.
reaction.
`
In view of the presence of the double bond in the compounds
described above, choosing 24 as a model compound, the easily
obtainable dibromo derivative 47 (and 48 derived therefrom) was
also synthesized from 24 (Table 1, last entry). As expected 47
obtained on addition of molecular bromine to the double bond in
24 was in the form of a pair of enantiomers (as also the derived 48)
as evident from the NMR spectra of 47. Further, in order to
establish the importance (or otherwise) of the sugar moiety in the
biological activity to be studied, a set of ether analogues 49 (benzyl
ether) and 50 (methyl ether) were also synthesized from methyl
ricinoleate 7. It must be pointed out that for the synthesis of the 12-
O-benzyl ricinoleate 49 from 7 and BnBr neither the method using
23
24
Ag2O nor the one using Cs2CO3 was successful. However, the
reaction of the two (7 and BnBr ) promoted by NaH yielded the
desired product 49 in admixture with 49a as an inseparable mixture
in a ratio of 1:0.5 as determined by the 1H NMR spectroscopy of the
chromatographically isolated mixture (see electronic SI for details).
The mixture was therefore subjected to the conditions of Zemplen
transesterification in MeOH whereby the benzyl ester 49a was
transformed into the methyl ester analogue 49 thus affording the
latter overall neatly.
Reagents and conditions: (i) HCl/MeOH, rt, 4 h, 90%; (ii) Ag2CO3, AgClO4,
powdered molecular sieves (4Å), CH2Cl2 rt, 10-12 h, 53-68%; (iii)
NaOMe/MeOH, 20-40 min, rt, 90-94%; (iv) LiOH/aq THF, 2-4 h, rt, 80-87%;
(v) Amberlite IR 120 (H+), rt, 80-88%
Scheme1. Synthesis of ricinoleic acid glycosides in their
partially/fully deprotected form and/as their sodium/lithium salts.
Table 1. Different ricinoleic acid glycosides and their partially or fully deprotected and sodium/lithium salt derivatives
Acetohalosugar
Glycosylated ricinoleates synthesized
Yield (%)
Structure
Compound number
used for synthesis
21, R1 = Ac, R2 = OCH3
22, R1 = H, R2 = OCH3
68
92
13
14
23, R1 = H, R2 = OH
87
58
25
24, R1 = Ac, R2 = OCH3
25, R1 = H, R2 = OCH3
26, R1 = H, R2 = OH
27, R1 = H, R2 = O-Li+
90
81
83
2 | J. Name., 2012, 00, 1-3
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28, R1 = Ac, R2 = OCH3
60
94
85
29, R1 = H, R2 = OCH3
30, R1 = H, R2 = OH
15
31, R1 = H, R2 = O-Na+
32, R1 = Ac, R2 = OCH3
80
52
33, R1 = H, R2 = OCH3
92
16
17
18
19
34, R1 = H, R2 = OH
83
66
35, R1 = Bz, R2 = OCH3
36, R1 = H, R2 = OCH3
91
37, R1 = H, R2 = OH
87
57
38, R1 = Bz, R2 = OCH3
39, R1 = H, R2 = OCH3
92
40, R1 = H, R2 = OH
79
60
41, R1 = Ac, R2 = OCH3
42, R1 = H, R2 = OCH3
94
43, R1 = H, R2 = OH
85
53
44, R1 = Ac, R2 = OCH3
45, R1 = H, R2 = OCH3
91
20
24
46, R1 = H, R2 = OH
84
80
47, R1 = Ac, R2 = OCH3
(For the structure,
see the entry at row
2 above)
48, R1 = H, R2 = OCH3
87
Evaluation of antimicrobial activity and observations on the
structure activity relationship (SAR)
Once in hand, the compounds described above were evaluated for
their antimicrobial activity. In the initial screening they were tested
for their activity against three Gram +ve bacteria, viz. Micrococcus
luteus (MTCC-2470), Staphylococcus aureus (MTCC-96), and Bacillus
subtilis (MTCC-121), three Gram –ve bacteria, viz. E. coli (MTCC-
739), Pseudomonas aeruginosa (MTCC-2453), Klebsiella planticola
(MTCC-530) and a fungus, Candida albicans (MTCC-3017). The
evaluations were done on the basis of the zone of inhibition (results
not shown here, see electronic SI for details) in the usual manner.
The deacylated glycosides of methyl ricinoleate 22, 25, 29, 36, 39,
42, 45, and 48 showed good inhibition of the Gram +ve bacteria
evaluated. However, the analogous N-acetyl-glucosamine-linked
methyl ricinoleate 33 was proved inactive. Also, the glycosides of
methyl ricinoleate showed better inhibition as compared to the
Likewise, the reaction of 7 with MeI (promoted by NaH) was also
successful in yielding the methyl ether analogue 50 but, again,
along with an inseparable by-product. But unlike 49a, the by-
product in the latter case was a mixture of 5026 and the lactone 50a
(in a ratio of 1:0.5) as characterized by 1H NMR spectroscopy (see
electronic SI for details).The separation of the lactone (50a) from
the ester (50)could however be very successfully facilitated by
methanolysis of the lactone (to yield 7) using anhydrous methanolic
hydrogen chloride followed by purification by column
chromatography.
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simple ether analogues methyl 12-O-benzyl ricinoleate (49) and (2 and 3) themselves. Based on these initial results compounds 22,
methyl 12-O-methyl ricinoleate (50). Neither methyl ricinoleate (7) 25, 29, 36,37,39, 42, 44, 45 and 48were taken up for the
nor ricinoleic acid/its lithium/sodium salt (1/3/2a) were also active determination of their MIC values against different Staphylococcal
against any of the strains tested above (see electronic SI for details). strains along with some of the standard drugs in use for
This showed the importance of the presence of sugar unit in the comparison. Of these glycosides studied 37, the L-rhamnosylated
structure for the antimicrobial activity. Smith et al while evaluating methyl ester, and 44 and 45,the lactosylated methyl esters, showed
the antimicrobial activity of 6-O-substituted fatty alkyl ethers/fatty high MIC values (>1000, results not shown) against the non-clinical
acyl esters of methyl glucopyranosides similarly had observed the S. aureus MTCC-96. This reiterated the fact that not only that the
importance of the presence of the sugar residue (as well as its acid has to be in its methyl ester form but also that not all sugars
nature) in the molecule for significant antimicrobial activity.27 are suitable as substituents on the ricinoleic acid derivatives for
Again, neither the acyl-protected glycosides of methyl ricinoleate them to be sufficiently biologically active. The above experiments
21, 24, 28, 32, 35, 38 and 41 nor the fully deprotected ricinoleic acid thus clearly elucidated the importance of the structure of the sugar
glycosides (as free acids) 23, 26, 30, 37, 40, 43 and 46 were active moiety present in the molecule. The poorer activity of the free acid
against either of the Gram +ve or the Gram –ve bacteria tested. 37 is in line with the fact that ester form of the acid is necessary for
However, compounds 37 (the rhamnosylated compound) and 44 the compound to be effectively active. Likewise, the bulky
(the lactose-linked compound) showed moderate inhibition against disaccharide units in 44 and 45 were also proven to be detrimental
Bacillus subtilis (MTCC-121). This revealed the importance of for good activity. Based on these results compounds 37, 44 and 45
ricinoleic acid present in the form of ester in imparting the were excluded from further in vitro screening experiments on the
inhibitory activity. In the work of Smith et al referred to above also, bacterial strains. Instead, for ascertaining the generality of
they had observed that the 6-O-acyl glucosides were more active application of these compounds to other Gram +ve bacterial strains,
than the corresponding ethers.27 The deacylated 9,10-dibromo Bacillus subtilis and Micrococcus luteus strains were included in the
compound 48 (as racemic mixture) showed better inhibition against study. The results obtained for the promising compounds chosen
M. luteus (MTCC-2470) and S. aureus (MTCC-96) compared to the are summarized in Table
2 and clearly demonstrates the
corresponding acylated compound 47 which was inactive. effectiveness of these compounds to act against all the Gram +ve
Moreover, the sodium/lithium salts of the deacylated ricinoleic acid bacteria tested.
glycosides 27 and 31 were also inactive as were the salts of the acid
Table 2 Antibacterial activity of methyl ricinoleate-glycosides against Staphylococcal, Bacillus subtilis and Micrococcus luteus strains
MIC (µg/ml)
S. aureus
MTCC-96 ~
ATCC 9144
(non-
clinical)
16
MRSA
831(clinical
isolate)
S. aureus
1199 (wild
type clinical
isolate)
S. aureus
1199B (Nor A subtilis (MTCC
overproducing
strain)
Bacillus
Micrococcus
luteus
(MTCC
Test
compounds/
Antibiotics
Entry
121 ~ ATCC
6051)
2470)
1
2
22
25
1000
1000
1000
1000
32
8
8
125
1000
32
16
2
16
2
8
3
29
2
2
0.25
0.125
0.5
1
0.5
0.5
1
4
36
4
250
8
1000
4
5
39
4
6
42
4
1000
1000
25
2
8
2
7
48
32
62.5
0.39
0.19
0.78
0.48
1
125
6.12
3.125
50
2
4
8
Erythromycin
Teicoplanin
Norfloxacin
Oxacillin
Ampicillin
Amoxicillin
Linezolid
Vancomycin
0.78
1.56
0.39
3.12
32
0.5
0.25
0.5
0.5
4
1
9
12.5
100
0.5
2
10
11
12
13
14
15
2000
250
1000
4
1
16
4
16
250
0.5
1.95
0.78
2
2
1.56
1.56
3.9
7.8
6.25
1
2
3.12
0.125
1
Possibly the glycosides of methyl ricinoleate would have possible that the glycolipids 29 and 39 may be playing some specific
permeabilized the bacterial cell membrane more effectively due to role in the bacterial cell wall biosynthesis due to the presence of
their better surfactant properties11 and thus showing the mannose and arabinose in them. They could also possibly act by
antibacterial activity. As it has been reported in the past, another inhibiting the transferases (or any other glycan-forming enzymes)
possible cause for the antibacterial properties of these compounds involved in the bacterial cell wall biosynthesis. To determine the
could be the inhibition of fatty acid biosynthesis (Fab1) enzyme above mentioned possible mechanism of action of these glycosides
which is an essential component of the fatty acid synthesis in Gram of ricinoleic acid a set of experiments with compounds 29, 39 and
+ve bacteria.11,28,29 Among the promisingly active compounds, 29 vancomycin (selectively active against Gram +ve bacteria, as the
and 39 showed (entries 3 and 5, Table 2) a wide spectrum positive control) was carried out as described below.
antibacterial activity against different Staphylococcal species. It is
4 | J. Name., 2012, 00, 1-3
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The binding of vancomycin to D-Ala-D-Ala prevents cell wall
synthesis of the long polymers of N-acetylmuramic acid and N-
acetylglucosamine that form the backbone strands of the bacterial
cell wall, and it prevents the backbone polymers that do manage to
form cross-linking with each other. Since the MIC of vancomycin is
more or less matching with 29 and 39, we used these compounds
for membrane permeability assay as well as cell membrane
disintegration assay (see below). The cell permeability effect of 29
900
800
700
600
500
400
300
200
100
0
Vancomycin
29
39
control
and 39 is comparable with vancomycin,
a membrane active
antibiotic. The uniform increase in the absorbance of o-nitrophenol
suggests that the non-membrane permeable o-nitrophenyl-β-
galactoside (ONPG) is slowly permeabilized inside the bacterial cells
and subsequently is hydrolyzed by the intracellular β-galactosidase
to o-nitrophenol that shows absorbance at 410 nm. As shown in Fig.
2 compounds 29 and 39 induced an increase in the permeability of
S. aureus cytoplasmic membrane over a period of time in a manner
comparable to that of vancomycin. Moreover, the test compounds
as well as vancomycin possessed similar MIC values against S.
aureus. This suggests that the cytoplasmic membrane would have
been permeabilized by compounds 29 and 39.
Time (in minutes)
Figure 3: Effect of 29 (squares), 39 (triangles) and vancomycin
(diamonds) on membrane integrity of S. aureus cells by propidium
iodide uptake assay. The untreated S. aureus cells (crosses) were
taken as control. For each sample 106 CFU ml-1 were analyzed. The
membrane integrity of S. aureus cells was destroyed by 29 and 39.
The experiment was carried out two times in triplicate. The results
were presented as mean ± SD
.
The cytoplasmic membrane is the main barrier that limits the
distribution and entry of antibiotics. In addition to the antimicrobial
activities, some serve as an anti-resistance compounds and are able
to interact with bacterial membranes, and create ion permeable
channels leading to an increased cytoplasmic membrane
permeability and hence, bacterial cell death.30 In the case of 29 and
39, they showed permeabilization effect on S. aureus membrane
and increased the plasma membrane permeability for entry of
ONPG into cells. Moreover, the gradual increase in the fluorescence
due to the influx of PI into the cells, indicates that the cytoplasmic
membrane could be the most probable target of action of ricinoleic
acid glycosides.
0.6
0.5
Vancomycin
0.4
0.3
0.2
0.1
0
29
39
Control
Time (H:MM:SS)
Cytotoxicity
Figure 2: The cytoplasmic membrane permeabilization of S. aureus
cells treated with compounds 29 (squares), 39 (triangles) and The compounds that showed good MIC values in the antibacterial
vancomycin (diamonds).The untreated S. aureus cells (crosses) activity evaluation were taken for the cell viability study with a view
were taken as control. (Compounds 29 and 39 induced an increase to assessing their toxicity characteristics. According to FDA castor
in the permeability of S. aureus. The experiment was carried out oil is classified as a safe and effective stimulant laxative18and it was
two times in triplicate. The results are presented as mean ± SD.)
hoped that attaching a sugar molecule would not result in seriously
altering its cytotoxic tolerance. It was therefore not surprising that
the synthesized glycosides of methyl ricinoleate 22, 25, 29, 33, 36,
39, 42, 45, and 48 exhibited no significant cytotoxicity when tested
up to 500 μg mL-1 (cell viability >75 %) on J774A.1 cells as shown in
Fig 4. However, at higher concentrations of 1000 μg mL-1
compounds 25, 42, 45, and 48demonstrated cell viability of only
56.2, 68.4, 67.5 and 56.8 % respectively. Thus, the results are
indicative of good potential for these compounds to be studied in
detail further
To verify whether the cells are permeabilized or disintegrated, the
propidium iodide (PI) uptake assay was carried out. PI is a viability
fluorescent marker that can penetrate impaired cells and
intercalate into nucleic acids. Thus, compounds 29-, 39- and
vancomycin- induced membrane damage of S. aureus cells, was
°
determined by treating the cells with PI at 37 C for half an hour
using Synergy reader (BioTek, USA). As shown in Fig. 3, in the
absence of any antibacterial agent, the untreated control cells of S.
aureus showed no PI fluorescence signal. However a significant
increase in the PI fluorescence signal was observed in the case of
29, 39 and vancomycin. These results indicate that the membrane
integrity of S. aureus cells was affected by the treatment with the
test compounds as comparable to that of vancomycin.
Figure 4: Cytotoxicity study of biologically active glycosides of
methyl ricinoleate (Results are expressed as mean ± SD of three
independent experiments performed in triplicates)
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For the initial screening of the antimicrobial activity exhibited by
synthesized compounds they were tested by the agar diffusion
method. We used Candida albicans MTCC 3017 and the battery of
Gram’s +ve and Gram -ve test bacterial strains, viz. Micrococcus
luteus MTCC 2470, Bacillus subtilis MTCC 121, Staphylococcus
aureus MTCC 96, E. coli MTCC 739, Pseudomonas aeruginosa MTCC
2453, Klebsiella planticola MTCC 530 with suitable positive control
for antibacterial and antifungal. The susceptibility of MRSA-831, SA-
1199, SA-1199B and SA-96 towards various antibiotics was studied.
It comprised MIC determination of antibiotics in Ca2+ and
Mg2+adjusted Muller Hinton Broth (MHB) as described previously
32.Based on literature, the specific concentration range was used for
individual antibiotic and serial dilutions were made accordingly.
Similarly, the MIC of standards and shortlisted test compounds was
determined in MHB. Briefly, the compounds were first dissolved in
DMSO and then diluted in MHB, to give the starting concentration
of 400 µg mL-1which was diluted across a 96 well microtiter plate in
two fold serial dilution to give the final concentration range from
400 to 0.195 µg mL-1. Bacterial inocula equivalent to the 0.5
McFarland standards were prepared in normal saline and diluted to
give the final density of 5×105 cfu mL-1. The inoculum (100 μL) was
added to all the wells and the microtiter plate was incubated at 37
°C for 48 h. The MIC was recorded as the lowest concentration at
which no bacterial growth was observed. This was facilitated by the
addition of 20 μL of MTT at the concentration 10 mg mL-1 in
methanol to each well and incubated at 37 °C for 20 min where
bacterial growth was indicated by purple coloration adhered to
cells. Appropriate DMSO, cell and sterile saline controls were
carried out in the same set of experiments.
Experimental
Materials and methods
All reagents and chemicals were purchased from Sigma-Aldrich and
were used without further purification. TLC analyses were
performed on 0.2 mm Merck pre-coated silica gel 60 F254 aluminium
sheets and the spots were visualized under UV lamp and/or by
immersion in an ethanolic solution of sulphuric acid (5%, v/v)
followed by heating. Final purifications were performed using silica
gel 200-400 mesh size. Specific rotations were recorded on a
Rudolph Autopol IV Polarimeter at 20 ˚C. NMR spectra were
recorded on a Bruker Avance DPX (400 MHz) spectrometer. 1H NMR
and 13C NMR spectra were referenced to the internal standard
tetramethylsilane, in the respective deuterated solvents. Coupling
constants (J) are reported in Hertz. All assignments were confirmed
with the aid of two-dimensional H-1H (COSY) and/or H-13C (HSQC)
experiments using standard pulse programs. Processing of the
spectra was performed with MestReNova software. High resolution
1
1
mass spectra (HRMS) were recorded on
a Bruker Maxis
spectrometer. Biosafety Cabinet (Clean Air, Chennai, India), CO2
incubator (WTC Binder, Tuttlingen, Germany), Ultracentrifuge
(Sigma, St. Louis, MO, USA), autopipettes, ELISA plate reader
(Labsystems, Helsinki, Finland) and Neubauer chamber (HBG,
Gießen, Germany) were used for the cell culture.
Materials and methods for antimicrobial evaluation
Chemicals and reagents used for biological evaluation
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
and standard antibiotics such as ampicillin, amoxicillin,
amphoterecin B, norfloxacin, oxacillin, linezolid, vancomycin,
teicoplanin and erythromycin were procured from Sigma-Aldrich,
USA. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine
serum (FBS), phosphate buffered saline (PBS), antibiotic solution
and other chemicals for the biological experiments were purchased
from Hi-media Limited (Mumbai, India). Lipopolysaccharide
(Escherichia coli 026:B6, LPS) and dimethyl sulfoxide (DMSO) were
purchased from Sigma Chemical Co. (St. Louis, MO, USA).
Cytoplasmic membrane permeability assay:
To demonstrate the membrane permeability, previously described
method was adopted.30,33 Staphylococcus aureus MTCC 96 cells
were grown in M9 minimal medium with lactose as a sole carbon
source from a single colony, overnight at 37°C. After three washings
in phosphate buffer saline (PBS, pH 7.4), the culture was diluted to
106 CFU ml-1 in PBS and added to all wells in a non-culture-treated
polystyrene microplate, together with compounds 29, 39 and
vancomycin (as a positive control) at MIC concentrations of 2µg ml-
1, 4 µg ml-1and 1.56 µg ml-1,respectively. Each well also contained
1.5mM ONPG in PBS. The plates were incubated with gentle shaking
at 37°C. The hydrolysis of ONPG to o-nitrophenol over time was
monitored at 410 nm with a microplate reader (BioTek, USA).
Similar procedure was adopted for untreated cells, taken as control.
Bacterial strains
Clinical isolate of S. aureus (MRSA-831) was obtained from
Government Medical College and Hospital, Chandigarh. S. aureus
1199 (SA-1199) is a methicillin- and fluoroquinolone-susceptible
clinical isolate. S. aureus 1199B (SA-1199B) is fluoroquinolone-
resistant mutant of SA-1199 strain that was recovered from the
blood and cardiac vegetation of rabbits that had experimental
endocarditis with SA-1199 and had failed ciprofloxacin therapy
given for the treatment of this infection.31Both the strains viz. SA-
1199 and SA-1199B were obtained from CSIR-Indian Institute of
Integrative Medicine, Jammu, India with kind permission of Dr G.
W. Kaatz, Wayne State University School of Medicine, and Detroit,
MI, USA. S. aureus MTCC 96 (SA-96), Micrococcus luteus MTCC
2470, Bacillus subtilis MTCC 121, E. coli MTCC 739, Pseudomonas
aeruginosa MTCC 2453, Klebsiella planticola 530 and Candida
albicans MTCC 3017 were obtained from Microbial Type Culture
Collection (MTCC), CSIR-Institute of Microbial Technology,
Chandigarh.
Propidium iodide uptake assay:
According to previously described method34 Staphylococcus aureus
MTCC 96 cells were collected in mid-log phase. This was followed by
addition of compounds 29, 39 and vancomycin (as a positive
control) at MIC concentrations of 2µg ml-1, 4µg ml-1 and 1.56µg ml-1,
respectively. Then the mixtures were incubated for 6 h at 3737°C.
The bacterial cells were washed 2-3 times with PBS (pH 7.4) and
resuspended at a concentration of 106 CFU ml-1 in PBS. Cells were
treated with propidium iodide (PI) at 10µM concentration and
aliquots of 200µl were transferred in triplicate to a 96-well plate.
With excitation wavelength of 535 nm and emission
wavelength of 625 nm, PI fluorescence was monitored for 30
min at 3 min interval using Synergy HT multi-mode microplate
reader (BioTek, USA). Similar procedure was adopted with
untreated cells, taken as control.
Susceptibility study of S. aureus strains and MIC determination
6 | J. Name., 2012, 00, 1-3
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Cell viability assay
General procedure for the preparation of ricinoleic acid-glycosides
as their Li+/Na+ salt
J774A.1 cells were obtained from the National Center for Cell
Science (NCCS, Pune, India) and were cultured in 250 ml culture Freshly prepared aqueous alkali (1M, LiOH or NaOH as desired; 0.3
flasks containing DMEM media supplemented with heat-inactivated mL/100 mg ester) was added to a solution of the methyl ricinoleate
10% FBS, 10,000 units ml-1 penicillin and 10 mg ml-1 streptomycin in derivative 24/28 in THF and was stirred for 3-5 h at room
0.9% saline, in a CO2 incubator (5% CO2 in air) at 37 °C. The temperature. The reaction mixture that turned turbid on the
conventional MTT assay was carried out to assess the cell viability addition of the alkali became clear by the completion of the
of J774A.1 cells using previously reported method.35 All test hydrolysis which was also confirmed by TLC. It was then
samples dissolved in DMSO and 0.05% DMSO was used as the concentrated to a small volume and was then diluted with EtOAc
control group. The cell viability was calculated in respect to that of when the product got precipitated. Filtration and successive
control (% of control).
washing with a small volume of cold water and EtOAc gave the
desired product 27/31 in good yields after drying
.
General experimental procedures
Lithium ricinoleate (3)
The title compound 3 was prepared from 2 (500 mg, 1.6 mmol),
THF (10 mL) and aqueous LiOH solution (1M, 1.5 mL) by the method
(4 h) described above in a yield of 83% (153 mg) as a white fluffy
solid. 1H NMR (400 MHz, DMSO) δ 5.43-5.33 (m, 2H, H-9, H-10)
3.47-3.43 (m, 1H, H-12), 2.09-2.06 (m, 2H, 2×H-11), 1.97 (q, 2H, J=
6.4 Hz, J= 6.5 Hz, 2×H-8), 1.90 (t, J= 7.4 Hz, 2×H-2) 1.46-1.23 (m,
20H, 2×H-7, 2×H-13 -(CH2)8-), 0.87 (t, J18,17 =6.8 Hz, 3H, -CH3); 13C
NMR (100 MHz, DMSO) δ 175.65 (C-1), 130.58 (C-9) 126.64 (C-10),
69.84 (C-12), 38.16 (C-2) 36.48 (C-11), 35.23 (C-8), 31.43, 29.45,
29.22, 29.11, 28.93, 28.84, 26.93, 26.30, 22.14, (m, -(CH2)10-), 14.02
(C-18); HR MS m/z Calculated for C18H33LiO3 [M+Na]+ = 327.2487,
found 327.2480.
General experimental procedure of Koenigs-Knorr glycosylation
conditions for the synthesis of glycosides of ricinoleic acid
Methyl ricinoleate (7, 2.5 equiv.) was added to a mixture of the
glycosyl halide (1336/1436/1536/1637/1738/1839/1940/2036), Ag2CO3
(1.1 equiv.), AgClO4(1.1 equiv.) and powdered molecular sieves (4Å,
weight equivalent to that of 7) in anhydrous CH2Cl2 and was stirred
at the ambient temperature (23-27 °C) for 14-18 h in the dark under
an inert gas atmosphere. When the reaction was complete (from
the completion of the consumption of 8 as judged by TLC) the
mixture was diluted with CH2Cl2 and the insolubles were separated
by filtration through a Celite-pad. The filtrate after concentration to
dryness under reduced pressure was chromatographed on a column
of silica gel using EtOAc:hexanes as the eluent to yield the
respective glycosides (21/24/28/32/35/38/41/44, respectively) of
methyl ricinoleate.
Methyl
12-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-
ricinoleate (21)
The title compound was prepared from 2,3,4,6-tetra-O-acetyl-α-D-
galactopyranosyl bromide (13, 1.00 g, 2.4 mmol) by the general
General experimental procedure for the de-acylation of acyl-
protected glycosides 21/24/28/32/35/38/41/44 by Zemplen’s
transesterification
procedure described above. The reaction time was 14 h; and 21
23
D
(1.05 g) was obtained in 68% yield as a thick syrup.
= ̶ 1.5° (c =
[α
]
1, CHCl3); 1H NMR (400 MHz, CDCl3) δ 5.46 – 5.38 (m, 1H, H-9′), 5.38
– 5.30 (m, 2H, H-4, H-10′), 5.19 (dd, J2,3= 10.5 Hz, J2,1 = 7.9 Hz, 1H, H-
2), 5.00 (dd, J3,2 = 10.5 Hz, J3,4 = 3.5 Hz, 1H, H-3), 4.50 (d, J1,2 = 7.9 Hz,
1H, H-1), 4.16 (dd, J6a,6b = 11.2 Hz, J6a,5 = 6.8 Hz, 1H, H-6a), 4.09 (dd,
J6b,6a = 11.2 Hz, J6b,5 = 6.8 Hz, 1H, H-6b), 3.88 (dt, J5,6a = J5,6b = 6.8 Hz,
J5,4 = 1.0 Hz, 1H, H-5), 3.65 (s, 3H, -OCH3), 3.59 – 3.47 (m, 1H, H-12′),
2.43 – 2.24 (m, 4H, , 2×H-11′, 2×H-2′ ), 2.13 (s, 3H, -OCOCH3 ), 2.02
(s, 3H, -OCOCH3), 2.02 (s, 3H, -OCOCH3), 2.00 – 1.95 (m, 5H, 2×H-8′,
-OCOCH3), 1.66 – 1.52 (m, 2H, 2×H-7′), 1.48 – 1.37 (m, 2H, 2×H-13′),
1.37 – 1.14 (m, 16H, -(CH2)8-), 0.86 (t, J18′ 17′ = 6.9 Hz, 3H, -CH3); 13C
NMR (100 MHz, CDCl3) δ 174.37 (C-1′), 170.48 , 170.46, 170.31,
169.36 (4×OCOCH3), 132.03 (C-9′), 125.08 (C-10′), 101.82 (C-1),
82.27 (C-12′), 71.21 (C-3), 70.61 (C-5), 69.34 (C-2), 67.20 (C-4), 61.46
(C-6), 51.54 (-OCH3), 34.19 (C-13′), 34.02 (C-2′), 33.41 (C-11′), 31.98,
29.62, 29.54, 29.29, 29.26, 29.22, 27.56, 25.31, 25.04, 22.80, 22.75
(m, -(CH2)10-), 20.88, 20.81, 20.76, 20.72 (4×OCOCH3), 14.18 (C-18′);
HR MS m/z Calculated for C33H54 O12 [M+Na]+ = 665.3513, found
665.3519.
To a solution of the glycoside 21/24/28/32/35/38/41/44/47 in
anhydrous MeOH was added a catalytic amount of NaOMe and it
was stirred at room temperature until the reaction was complete
(0.5-4 h, depending upon the sugar derivative used, as judged by
TLC). The solution was neutralized with Amberlite IR 120 H+ resin
and, after the removal of the used resin by filtration, was
concentrated to afford the respective deacylated product
(22/25/29/33/36/39/42/45/48) in good yield. In all cases they were
obtained in the form of syrup except 33 which was obtained as an
amorphous fluffy powder.
General procedure for the saponification of methyl ricinoleate-
glycosides 21/24/28/32/35/38/41/44
Freshly prepared aqueous LiOH (1M, 0.3 mL/100 mg ester,
21/24/28/32/35/38/41/44) was added to the desired acyl-
protected glycoside of the methyl ricinoleate dissolved in THF (2
mL/100 mg) and was allowed to stand with gentle stirring for 3-8 h
at room temperature. The reaction mixture that turned turbid on
the addition of the alkali became clear by the completion of the
hydrolysis which was also confirmed by TLC. Addition of Amberlite
IR 120 H+ resin to a slightly acidic pH (approximately 6) followed by
removal of the used resin by filtration and concentration of the
filtrate under reduced pressure to dryness afforded the respective
ricinoleic acids 23/26/30/34/37/40/43/46in good yield.
Methyl 12-O-(β-D-galactopyranosyl)-ricinoleate (22)
The title compound was prepared from 21 (200 mg, 0.31 mmol) by
the general procedure described above. The reaction time was 0.5
23
D
h; and 22 (135 mg) was obtained in 92% yield as a thick syrup.
[α
]
1
= ̶ 9.8° (c = 1, CHCl3); H NMR (400 MHz, CD3OD) δ 5.52 – 5.39 (m,
2H, H-9′, H-10′), 4.31 (d, J1,2 = 7.1 Hz, 1H, H-1), 3.87 (d, J4,3 = J4,5 = 2.8
Hz, 1H, H-4), 3.80 – 3.69 (m, 3H, H-6a, 6b, H-12′), 3.67 (s, 3H, OCH3),
3.56 – 3.45 (m, 3H, H-2, H-3, H-5), 2.49 – 2.28 (m, 4H, 2×H-11′, 2×H-
This journal is © The Royal Society of Chemistry 20xx
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8′), 2.14 – 2.00 (m, 2H, 2×H-2′), 1.67 – 1.58 (m, 2H, H-7′), 1.58 – 1.50 (4×OCOCH3), 14.09 (C-18′); MALDI MS m/z Calculated for C33H54 O12
(m, 2H, 2×H-13′), 1.50 – 1.27 (m, 16H, -(CH2)8-), 0.92 (t, J18′ 17′ = 6.6 [M+K]+ = 681.873, found 681.747.
Hz 3H, -CH3); 13C NMR (100 MHz, CD3OD) δ 175.99 (C-1′), 132.27 (C-
9′), 126.95 (C-10′), 104.81 (C-1), 81.10 (C-12′), 76.35 (C-3), 75.04 (C- Methyl 12-O-(β-D-glucopyranosyl)-ricinoleate (25)
2), 72.78 (C-5), 70.12 (C-4), 62.22 (C-6), 51.98 (-OCH3), 34.80 (C-13′), The title compound was prepared from 24 (150 mg, 0.23 mmol)
34.23 (C-8′), 33.02 (C-11′), 30.67, 30.29, 30.22, 30.17, 28.41, 26.14, following the general procedure described above. The reaction time
26.02, 23.72 (m, -(CH2)10-), 14.47 (C-18′); HR MS m/z Calculated for was 0.5 h; and 25 (98 mg was obtained in 90% yield as a thick syrup.
1
C25H46 O8 [M+Na]+ = 497.3090, found 497.3110.
=
̶
21.4° (c = 0.8, CHCl3); H NMR (400 MHz, CD3OD) δ 5.51 –
23
D
[α
]
5.40 (m, 2H, H-9′, H-10′), 4.34 (d, J1,2= 7.8 Hz, 1H, H-1), 3.86 (dd,
J6a,6b= 11.8 Hz, J6a,5 = 2.3 Hz, 1H, H-6a), 3.75 – 3.67 (m, 2H, H-12′, H-
6b), 3.66 (s, 3H, -OCH3), 3.39 -3.34 (m, 2H, H-3, H-4), 3.28 – 3.23 (m,
1H, H-5), 3.18 (t, J1,2= J2,3= 8.4 Hz, 1H, H-2), 2.49 – 2.26 (m, 4H, 2×H-
11′, 2×H-2′), 2.13 – 2.02 (m, 2H, 2×H-8′), 1.67 – 1.58 (m, 2H, 2×H-7′),
12-O-(β-D-Galactopyranosyl)-ricinoleic acid (23)
The title compound was prepared from 21 (300 mg, 0.46 mmol) by
the general procedure described above. The reaction time was 0.5
h; and 23 (185 mg) was obtained in 87% yield as a thick syrup.
= ̶
9.9° (c = 0.3, MeOH); 1H NMR (400 MHz, DMSO) δ 5.37 (m, 2H, H- 1.57 – 1.44 (m, 2H, 2×H-13′), 1.44 – 1.22 (m, 16H, -(CH2)8- ), 0.91 (t,
23
D
[α
]
9′, H-10′), 4.11 (d, J1,2 = 4.5 Hz, 1H, H-1), 3.85 – 3.33 (m, 4H, H-4, H- J18′ 17′ = 6.8Hz, 3H, -CH3); 13C NMR (100 MHz, CD3OD) δ 176.01 (C-1′),
12′, H-6a,b), 3.33 – 3.16 (m, 3H, H-5, H-3, H-2), 2.38 – 2.07 (m, 4H, 132.35 (C-9′), 126.89 (C-10′), 104.20 (C-1), 81.14 (C-12′), 78.14 (C-3),
2×H-11′, 2×H-2′), 1.97 (m, 2H, 2×H-8′), 1.56 – 1.03 (m, 20H, -(CH2)10- 77.76 (C-5), 75.34 (C-2), 71.69 (C-4), 62.82 (C-6), 51.98 (-OCH3),
), 0.84 (t, J18′,17′ = 6.6 Hz, 3H, CH3); 13C NMR (100 MHz, DMSO) δ 34.80 (C-13′), 34.79 (C-2′), 34.20 (C-11′), 33.03, 30.69, 30.66, 30.30,
174.83 (C-1′), 131.11 (C-9′), 126.18 (C-10′), 103.72 (C-1), 78.92 (C- 30.24, 30.18, 28.42, 26.14, 26.02, 23.72 (m, -(CH2)10- ) 14.46 (C-18′);
12′), 75.04 (C-5), 73.68 (C-2), 70.96 (C-3), 68.12 (C-4), 60.38 (C-6), HR MS m/z Calculated for C25H46 O8 [M+Na]+ = 497.3090, found
33.87 (C-2′), 33.51 (C-11′), 33.15 (C-13′), 31.51, 29.21, 29.15, 28.86, 497.3098.
28.77, 28.75, 27.04, 24.69, 24.54, 22.28 (m, -(CH2)8-) 14.19 (C-18′);
HR MS m/z Calculated for C24H44 O8 [M+Na]+ = 483.2934, found 12-O-(β-D-Glucopyranosyl)-ricinoleic acid (26)
483.2941.
The title compound was prepared from 24 (250 mg, 0.39 mmol)
following the general procedure described above. Reaction time
Methyl
ricinoleate (24)
12-O-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)- was 3 h; and 26 (145 mg) was obtained in 81% yield as a thick syrup.
1
23
=
̶
14.8° (c = 0.3, MeOH); H NMR (400 MHz, CD3OD) δ 5.51 –
[α
]
D
The title compound was prepared from 2,3,4,6-tetra-O-acetyl-α-D-
glucopyranosyl bromide (14, 500 mg, 1.2 mmol) by the general
procedure described above. The reaction time was 14 h; and 24
5.35 (m, 2H, H-9′, H-10′ ), 4.32 (d, J1,2 = 7.8 Hz, 1H, H-1), 3.84 (dd,
J6a,6b= 11.8 Hz, J6a,5 = 2.2 Hz, 1H, H-6a), 3.74 – 3.61 (m, 2H, H-12′, H-
6b), 3.38 – 3.30 (m, 2H, H-3, H-4), 3.27 – 3.21 (m, 1H, H-5), 3.16 (t,
J1,2= J2,3= 8.3 Hz, 1H, H-2), 2.47 – 2.22 (m, 4H, m, 4H, 2×H-11′, 2×H-2′
23
D
(455 mg) was obtained in 58% yield as a thick syrup.
= +10.1° (c
[α
]
1
= 0.5, CHCl3); H NMR (400 MHz, CDCl3) δ 5.47 – 5.38 (m, 1H, H-9′), ), 2.10 – 1.99 (m, 2H, 2×H-8′), 1.64 – 1.55 (m, 2H, 2×H-7′ ), 1.55 –
5.35 (m, 1H, H-10′), 5.19 (t, J3,2 = J3,4 = 9.5 Hz, 1H, H-3), 5.06 (t, J4,5
=
1.47 (m, 2H, 2×H-13′), 1.48 – 1.24 (m, 16H, -(CH2)8-), 0.89 (t, J18′ 17′ =
J4,3 = 9.7 Hz, 1H, H-4), 4.97 (dd, J2,3 = 9.6 Hz, J2,1 = 8.0 Hz, 1H, H-2), 6.6 Hz, 3H, -CH3); 13C NMR (100 MHz, CD3OD) δ 178.05 (C-1′),
4.55 (d, J1,2 = 8.0 Hz, 1H, H-1), 4.24 (dd, J6a,6b = 12.2 Hz, J6a,5 = 5.2 Hz, 132.37 (C-9′), 126.86 (C-10′), 104.17 (C-1), 81.13 (C-12′), 78.11 (C-3),
1H, H-6a), 4.11 (dd, J6b,6a = 12.1 Hz, J6b,5 = 2.5 Hz, 1H, H-6b), 3.72 – 77.72 (C-5), 75.31 (C-2), 71.67 (C-4), 62.80 (C-6), 35.26 (C-13′), 34.77
3.66 (m, 1H, H-5), 3.66 (s, 3H, -OCH3), 3.59 – 3.49 (m, 1H, H-12′), (C-2′), 34.18 (C-11′), 32.92, 30.71, 30.65, 30.35, 30.27, 28.43, 26.20,
2.47 – 2.18 (m, 4H, 2×H-11′, 2×H-2′), 2.12 – 1.92 (m, 14H, 26.12, 23.71 (m, -(CH2)10-), 14.47 (C-18′); HR MS m/z Calculated for
4×OCOCH3, 2×H-8′), 1.67 – 1.53 (m, 2H, 2×H-7′), 1.51 – 1.36 (m, 2H, C24H44 O8 [M+Na]+ = 483.2934, found 483.2934.
2×H-13′), 1.38 – 1.11 (m, 16H, -(CH2)8-), 0.87 (t, J18′ 17′= 6.9 Hz, 3H, -
CH3 ); 13C NMR (100 MHz, CDCl3) δ 174.44 (C-1′), 170.80, 170.53, Lithium 12-O-(β-D-glucopyranosyl)-ricinoleate (27)
169.57, 169.34 (4×OCOCH3), 132.09 (C-9′), 125.06 (C-10′), 101.22 (C- The title compound was prepared from 24 (250 mg, 0.39 mmol) by
1′), 82.10 (C-12′), 73.11 (C-3), 71.78 (C-2), 71.72 (C-5), 68.76 (C-4), the general procedure described above using LiOH as the alkali. The
62.34 (C-6), 51.59 (-OCH3), 34.22 (C-13′), 34.03 (C-2′), 33.35 (C-11′), reaction time was 3 h; and 27 was obtained as colourless fluffy solid
23
D
32.00, 29.65, 29.54, 29.32, 29.28, 29.25, 27.57, 25.31, 25.07, 22.77 (152 mg) in 83% yield.
= ̶
8.4° (c = 0.5, H2O); 1H NMR (400 MHz,
[α
]
(m, -(CH2)8-), 20.86, 20.80, 20.79, 20.76 (4×OCOCH3), 14.21 (C-18′);
D2O) δ 5.56 – 5.32 (m, 2H, H-9′, H-10′), 4.37 (d, J1,2 = 7.7 Hz, 1H, H-
1), 3.86 – 3.72 (m, 2H, H-6a, 6b), 3.71 – 3.58 (m, 1H, H-12′), 3.51 –
3.39 (m, 2H, H-3, H-4), 3.34 – 3.18 (m, 2H, H-5, H-2), 2.53 – 2.20 (m,
2H, 2×H-11′), 2.14 (t, J2′,3′ = 7.5 Hz 2H, 2×H-2′), 2.08 – 1.95 (m, 2H,
2×H-8′), 1.59-1.45 (m, 4H, 2×H-7′, 2×H-13′), 1.41-1.18 (m, 16H, -
(CH2)8-) 0.84 (t, J18′,17′ = 6.5 Hz, 3H, -CH3); 13C NMR (100 MHz, D2O) δ
181.50 (C-1′), 132.55 (C-9′), 125.06 (C-10′), 102.55 (C-1), 80.89 (C-
12′), 75.96 (C-3), 75.71 (C-5), 73.23 (C-2), 69.27 (C-4), 60.54 (C-6),
37.81 (C-2′), 33.31 (C-13′), 32.61 (C-11′), 31.60, 29.32, 29.28, 29.14,
29.01, 28.95, 27.23, 26.14, 24.84, 23.24, 22.48 (m, -(CH2)10-),
HR MS m/z Calculated for C33H54 O12 [M+Na]+ = 665.3513, found
665.3519.
The orthoester 9a formed as the by-product in the above reaction
was also isolated (132 mg, 17 %). 1H NMR (400 MHz, CDCl3) δ 5.59 –
5.45 (m, 1H, H-9′), 5.42 – 5.30 (m, 1H, H-10′), 5.18 (t, J3,4 = J3,2 = 9.7
Hz, 1H, H-3), 5.02 (d, J1,2 =4.0 Hz, 1H, H-1), 4.99 (d, J4,3 = J4,5 = 9.8 Hz,
1H, H-4), 4.26 (dd, J6a,6b= 12.3 Hz, J6a,5 = 4.8 Hz, 1H H-6a), 4.11 – 4.00
(m, 2H, H-6b, H-5), 3.71 – 3.59 (m, 5H, H-2, H-12′, -OCH3), 2.38 –
2.22 (m, 4H, 2×H-11′, 2×H-2′ ), 2.13 – 1.98 (m, 11H, 3×OCOCH3, 2×H-
8′), 1.70 – 1.47 (m, 7H, 2×H-7′, 2×H-13′, -CH3), 1.44 – 1.14 (m, 16H, -
(CH2)8-), 0.89 (t, J18′ 17′ = 7.0 Hz, 3H, -CH3); 13C NMR (100 MHz, CDCl3)
δ 174.34 (C-1′), 170.97, 170.69, 169.67 (3×OCOCH3), 133.28 (C-9′),
124.64 (C-10′), 97.84 (C-1), 79.95 (C-12′), 77.23 (O-C-O), 73.51 (C-3),
70.95 (C-2) , 68.06 (m, C-4, C-5), 62.11 (C-6), 51.48 (-OCH3), 34.76 (-
CH3), 34.10 (C-13′), 31.79 (C-2′), 31.45 (C-11′), 29.42, 29.38, 29.13,
29.10, 27.39, 25.62, 24.94, 22.63 (m, -(CH2)8- ), 20.93, 20.74, 20.69
13.89(C-18′); HR MS m/z Calculated for C24H43LiO8 [M+Na]+
489.3016, found 489.3011.
=
Methyl
ricinoleate (28)
12-O-(2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyl)-
8 | J. Name., 2012, 00, 1-3
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ARTICLE
The title compound was prepared from 2,3,4,6-tetra-O-acetyl-α-D- 2×H-7′, 2×H-13′), 1.46 – 1.14 (m, 16H, -(CH2)8-), 0.87 (t, J17′,18′ = 6.3
mannopyranosyl bromide (15, 1.00 g, 2.4 mmol) following the Hz, 3H, -CH3); 13C NMR (100 MHz, D2O) δ 183.42 (C-1′), 132.82 (C-
general procedure described above. The reaction time was 14 h; 9′), 124.39 (C-10′), 98.36 (C-1), 76.54 (C-12′), 72.95 (C-4), 70.81 (C-
23
D
and 28 (0.92 mg) was obtained in 60% yield as a thick syrup.
=
2), 70.76 (C-3), 66.10 (C-5), 60.43 (C-6), 37.71 (C-2′), 34.14 (C-13′),
31.54, 30.43, 29.31, 29.23, 29.14, 28.99, 28.91, 27.20, 26.11, 25.38,
22.49 (m, -(CH2)11-), 13.85 (C-18′); HR MS m/z Calculated for
C24H43NaO8 [M+Na]+ = 505.2753, found 505.2751.
[α
]
1
+51.4° (c = 0.5, CHCl3); H NMR (400 MHz, CDCl3) δ 5.53 – 5.41 (m,
1H, H-9′), 5.38 – 5.21 (m, 3H, H-3, H-10′ H-4), 5.17 (dd, J2,3 = 3.3 Hz,
J2,1 = 1.8 Hz, 1H, H-2), 4.94 (d, J1,2= 1.6 Hz, 1H, H-1), 4.27 (dd, J6a,6b
=
12.7 Hz, J6a,5 = 5.8 Hz, 1H, H-6a), 4.11 – 4.02 (m, 2H, H-6b, H-5), 3.66
(s, 3H, -OCH3), 3.65 – 3.60 (m, 1H, H-12′), 2.33 – 2.19 (m, 4H, 2×H-2′,
2×H-11′), 2.15 (s, 3H, -OCOCH3), 2.09 (s, 3H, -OCOCH3), 2.04 (s, 3H, -
OCOCH3), 2.02 – 1.97 (m, 5H, -OCOCH3, 2×H-8′), 1.67 – 1.46 (m, 4H,
Methyl
12-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-
glucopyranosyl)-ricinoleate (32)
The title compound was prepared from 2-acetamido-3,4,6-tri-O-
acetyl-2-deoxy-α-D-glucopyranosyl chloride (16, 1.00 g, 2.74 mmol)
following the general procedure described above. The reaction time
4H, 2×H-7′, 2×H-13′), 1.42 – 1.18 (m, 16H, -(CH2)8- ), 0.88 (t, J18′,17′
=
6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 174.43 (C-1′), 170.77,
170.19, 170.00, 169.91 (4×OCOCH3), 132.85 (C-9′), 124.32 (C-10′),
96.48 (C-1), 78.81 (C-12′), 70.30 (C-2), 69.30 (C-3), 68.91 (C-5), 66.43
was 16 h; and 32 (930 mg) was obtained as a colorless glassy solid in
= ̶
15.4° (c = 0.5, CHCl3); 1H NMR (400 MHz, CDCl3) δ 5.61
23
D
53%).
[α
]
(C-4), 62.74 (C-6), 51.57 (-OCH3), 34.52 (C-13′), 34.24 (C-8′), 31.91, (brs, 1H, -NH), 5.47 – 5.28 (m, 3H, H-9′, H-10′, H-3), 5.02 (t, J4,3 = J4,5
31.34, 29.61, 29.45, 29.29, 29.25, 27.52, 25.83, 25.08, 22.76, 21.08, = 9.6 Hz, 1H, H-4), 4.78 (d, J1,2 = 8.3 Hz, 1H, H-1), 4.23 (dd, J6a,6b
=
20.87, 20.85 (m, -(CH2)10-), 14.22 ( C-18′); HR MS m/z Calculated for 12.1 Hz, J6a,5= 5.2 Hz, 1H, H-6a), 4.10 (dd, J6b,6a= 12.1 Hz, J6b,5= 1.9
C33H54 O12 [M+Na]+ = 665.3513, found 665.3550.
Hz, 1H), 3.78 – 3.67 (m, 2H, H-2, H-5), 3.65 (s, 3H, -OCH3), 3.57 –
3.46 (m, 1H, H-12′), 2.43-2.26 (m, 4H, 2×H-11′, 2×H-2′), 2.07- 1.89
(m, 14H, 4×OCOCH3, 2×H-8′), 1.67 – 1.53 (m, 2H, 2×H-7′), 1.48 –
Methyl 12-O-(α-D-mannopyranosyl)-ricinoleate (29)
The title compound was prepared from 28 (0.5 g, 0.78 mmol) by the 1.37 (m, 2H, 2×H-13′), 1.26 (m, 16H, -(CH2)8-) 0.86 (t, J18′,17′ = 6.8 Hz,
general procedure described above. The reaction time was 0.5 h; 3H, -CH3); 13C NMR (100 MHz, CDCl3) δ 174.53 (C-1′), 170.99,
and compound 29 (0.35 g) was obtained in 94% yield as a thick 170.84, 170.14, 169.64 (4×OCOCH3), 132.03 (C-9′), 125.22 (C-10′),
1
23
D
syrup.
= +49.8° (c = 0.5, CHCl3); H NMR (400 MHz, CD3OD) δ 101.15 (C-1), 82.11 (C-12′), 72.42 (C-3), 71.69 (C-5), 69.08 (C-4),
62.56 (C-6), 55.66 (C-2), 51.61 (-OCH3), 34.22 (C-13′), 34.05 (C-2′),
[α
]
5.56 – 5.45 (m, 1H, H-9′), 5.45 – 5.36 (m, 1H, H-10′), 4.92 (s, 1H, H-1)
3.84 – 3.59 (m, 10H, H-2, H-6a,b, H-12′ H-3, H-4, H-5, -OCH3), 2.39 –
2.23 (m, 4H, 2×H-2′, 2×H-11′), 2.13 – 2.03 (m, 2H, 2×H-8′), 1.68 –
1.57 (m, 2H, 2×H-7′), 1.58 – 1.49 (m, 2H, H-13′), 1.47 – 1.26 (m,
16H), 0.92 (t, J17′,18′ = 6.5 Hz, 3H, -CH3); 13C NMR (100 MHz, CD3OD)
δ 175.98 (C-1′), 133.03 (C-9′), 126.07 (C-10′), 99.91 (C-1), 77.59 (C-
12′), 74.84 (C-4), 72.67 (C-2), 72.64 (C-3), 68.45 (C-5), 62.79 (C-6),
51.98 (-OCH3), 35.68 (C-13′), 34.79 (C-11′), 32.98, 31.82, 30.64,
30.54, 30.26, 30.21, 30.15, 28.37, 26.69, 26.01, 23.73 (m, -(CH2)10-),
33.35 (C-11′), 32.05, 29.61, 29.56, 29.29, 29.23 (m, -(CH2)8-), 27.54
(C-8′), 25.50 (-(CH2)8-), 25.06 (C-7′), 23.46 (NHCOCH3), 22.79 , 20.87,
20.81 (3×OCOCH3), 14.22 (C-18′); HR MS m/z Calculated for
C33H55NO12 [M+Na]+ = 664.3673, found 664.3694.
Methyl
12-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-
ricinoleate (33)
The title compound was prepared from 32 (0.5 g, 0.78 mmol)
following the general procedure described above. The reaction time
was 0.5 h; 16 h; and 33 (0.46 g) was obtained as a light brown glassy
14.48 (C-18′); HR MS m/z Calculated for C25H46 O8 [M+Na]+
497.3091, found 497.3091.
=
1
23
solid in 92% yield.
=
̶
40.8° (c = 0.5, CHCl3); H NMR (400 MHz,
[α
]
D
12-O-(α-D-Mannopyranosyl)-ricinoleic acid (30)
CD3OD) δ 5.41 (m, 2H, H-9′, H-10′ ), 4.49 (d, J1,2 = 8.2 Hz, 1H, H-1),
The title compound was prepared from 28 (300 mg, 0.46 mmol) by 3.86 (d, J6a,6b= J6a,5= 11.8 Hz, 1H, H-6a), 3.70 (dd, J6b,6a= 11.8 Hz,
the general procedure described above. The reaction time was 3 h; J6b,5= 5.1 Hz, 1H, H-6b), 3.65 (s, 3H, -OCH3), 3.60 (m, 2H, H-12′, H-2),
and compound 30 (180 mg) was obtained as a thick syrup in a yield 3.49 (t, J3,2 = J3,4 = 9.3 Hz, 1H, H-3), 3.38 – 3.22 (m, 2H, H-4, H-5),
1
23
D
of 85%).
= +68.4° (c = 0.5, MeOH); H NMR (400 MHz, DMSO) δ 2.56-2.20 (m, 4H, 2×H-11′, 2×H-2′), 2.05 (m, 2H, 2×H-8′), 1.97 (s, 3H,
NHCOCH3), 1.66 – 1.54 (m, 2H, 2×H-7′), 1.51 – 1.39 (m, 2H, 2×H-13′),
[α
]
5.50 – 5.21 (m, 2H, H-9′, H-10′), 4.73 (s, 1H, H-1), 3.85 – 3.00 (m, 7H,
H-2, H-3, H-4, H-5, H-6a,b, H-12′), 2.18 (m, 4H, 2×H-11′, 2×H-2′),
1.99 (m, 2H, 2×H-8′), 1.58 – 1.01 (m, 20H, -(CH2)10-), 0.85 (t, J18′,17′
1.41-1.29 (m, 16H, -(CH2)8-), 0.90 (t, J18′,17′ = 6.2 Hz, 3H, CH3); 13C
NMR (100 MHz, CD3OD) δ 176.17 (NHCOCH3), 173.53 (C-1′), 132.52
(C-9′), 126.70 (C-10′), 103.01 (C-1), 82.31 (C-12′), 77.71 (C-5), 75.83
(C-3), 72.00 (C-4), 62.74 (C-6), 57.85 (C-2), 52.06 (-OCH3), 34.81 (C-
2′), 34.21 (C-13′), 33.10 (C-11′), 30.72, 30.67, 30.28, 30.22, 30.16,
28.40, 26.17, 26.01, 23.76 (m, -(CH2)8-) 23.16 (NHCOCH3), 14.48 (C-
18′); HR MS m/z Calculated for C27H49NO8 [M+Na]+ = 538.3356,
found 538.3375.
=
6.6 Hz, 3H, CH3); 13C NMR (100 MHz, DMSO) δ 174.63 (C-1′), 131.61
(C-9′), 125.18 (C-10′), 97.89 (C-1), 74.38 (C-12′), 74.31 (C-4), 71.04
(C-2), 70.89 (C-3), 66.85 (C-5), 61.23 (C-6), 33.99 ( C-2′), 33.73 (C-
11′), 31.36 (C-13′), 30.22, 29.08, 28.87, 28.74, 28.66, 28.62, 26.90,
25.06, 24.57, 22.18 (m, -(CH2)10-) 14.06 (C-18′); HR MS m/z
Calculated for C24H44 O8 [M+Na]+ = 483.2934, found 483.2938.
12-O-(2-Acetamido-2-deoxy-β-D-glucopyranosyl)-ricinoleic
(34)
acid
Sodium 12-O-(α-D-mannopyranosyl)-ricinoleate (31)
The title compound was prepared from 28 (200 mg, 0.31 mmol) by
the general procedure described above using NaOH as the alkali.
The title compound was prepared from 32 (200 mg, 0.31 mmol) by
the general procedure described above. The reaction time was 3 h;
and compound 34 (130 mg) was obtained as a light brown fluffy
The reaction time was 3 h; and compound 31 (120 mg) was
23
D
obtained in 80% yield as a colourless fluffy solid.
= +45.9° (c =
[α
]
1
23
D
solid in 83% yield.
= +4.8° (c = 0.25, MeOH); H NMR (400 MHz,
1
[α
]
0.5, H2O); H NMR (400 MHz, D2O) δ 5.62 – 5.46 (m, 1H, H-9′), 5.46
– 5.28 (m, 1H, H-10′), 4.98 (s, 1H, H-1), 3.90 – 3.82 (m, 2H, H-2, H-
6a), 3.82 – 3.57 (m, 5H, H-3, H-6a, H-12′, H-5, H-4), 2.37 – 2.12 (m,
4H, 2×H-11′, 2×H-2′), 2.12 – 1.99 (m, 2H, 2×H-8′), 1.69 – 1.46 (m, 4H,
DMSO) δ 7.86 (d, JNH,2 = 7.2 Hz, 1H, -NH), 5.43 – 5.26 (m, 2H, H-9′, H-
10′), 4.31 (d, J1,2 = 6.9 Hz, 1H, H-1), 3.72 – 3.51 (m, 2H, H-6a,b), 3.46
– 3.19 (m, 3H, H-12′, H-2, H-5), 3.17 – 2.83 (m, 2H, H-3, H-4), 2.46 –
This journal is © The Royal Society of Chemistry 20xx
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2.01 (m, 2H, 2×H-11′), 2.03 – 1.80 (m, 4H, 2×H-8′, 2×H-2′), 1.76 (s, 2×H-11′, 2×H-2′), 2.14 – 1.98 (m, 2H, 2×H-8′), 1.65 – 1.45 (m, 4H,
3H, NHCOCH3), 1.43 – 1.00 (m, 20H, -(CH2)10-), 0.84 (t, J18′,17′ = 6.6 2×H-7′, 2×H-13′), 1.45 – 1.26 (m, 16H, -(CH2)8-), 1.24 (d, J5,CH3 = 6.2
Hz, 3H, CH3); 13C NMR (100 MHz, DMSO) δ 168.97 (C-1′), 131.24 (C- Hz, 3H, -CH3), 0.90 (t, J17′,18′ = 6.7 Hz, 3H, -CH3); 13C NMR (100 MHz,
9′), 125.90 (C-10′), 102.13 (C-1), 80.25 (C-12′), 76.95 (C-4), 74.04 (C- CD3OD) δ 177.78 (C-1′), 132.80 (C-9′), 126.69 (C-10′), 101.09 (C-1),
5), 70.81 (C-3), 61.24 (C-6), 55.91 (C-2), 33.44, 33.11, 31.45, 29.45, 79.40 (C-12′), 73.92 (C-4), 72.69 (C-2), 72.44 (C-3), 70.02 (C-5), 34.98
29.22, 29.09, 29.07, 28.86, 26.97, 24.42, 23.10, 22.20 (m, -(CH2)8-), (C-2′), 34.38 (C-13′), 33.72 (C-11′), 32.97, 30.63, 30.53, 30.28, 30.22,
14.08 (C-18′); HR MS m/z Calculated for C26H47NO8 [M+Na]+
524.3200, found 524.3193.
=
30.19, 28.36, 26.14, 26.08, 23.65 (m, -(CH2)10), 17.92 (-CH3), 14.44
(C-18′): HR MS m/z Calculated for C24H44 O7 [M+Na]+ = 467.2985,
found 467.2985.
Methyl
12-O-(2,3,4-tri-O-benzoyl-α-L-rhamnopyranosyl)-
ricinoleate (35)
Methyl
12-O-(2,3,5-tri-O-benzoyl-β-D-arabinofuranosyl)-
The title compound was prepared from 2,3,4-tri-O-benzoyl-α-L- ricinoleate (38)
rhamnopyranosyl bromide (17, 1.00 g, 2.0 mmol) following the The title compound was prepared from 2,3,5-tetra-O-benzoyl-α-D-
general procedure described above. The reaction time was 18 h; arabinofuranosyl chloride (18,2.18 g, 4.5 mmol) by the general
and compound 35 (1.016 g) was obtained in 66% yield as a thick procedure described above. The reaction time was 16 h; and 38
1
23
D
23
D
syrup.
= +84.4° (c = 0.5, CHCl3); H NMR (400 MHz, CDCl3) δ 8.11 (1.95 g) was obtained in a yield of 57% as a thick syrup.
=
̶
[α
]
[α
]
(dd, JA,B = 8.0 Hz, JA,B = 1.0 Hz, 2H, Ph-CH), 7.98 (dd, JA,B = 8.1, JA,B
=
14.4° (c = 0.5, CHCl3); 1H NMR (400 MHz, CDCl3) δ 8.14 – 7.94 (m,
1.0 Hz, 2H, Ph-CH ), 7.83 (dd, JA,B = 8.2 Hz, JA,B = 1.0 Hz, 2H, Ph-CH), 6H, Ph-CH), 7.63 – 7.27 (m, 9H, Ph-CH), 5.62 – 5.26 (m, 5H, H-3, H-2,
7.65 – 7.55 (m, 1H, Ph-CH ), 7.55 – 7.35 (m, 6H, Ph-CH), 7.26 (dd, JA,B H-9′, H-10′, H-1), 4.80 (d, J5a,5b= J5a,4= 10.8 Hz, 1H, H-5a), 4.73 – 4.53
= 9.0 Hz, JA,B = 6.6 Hz, 2H, Ph-CH), 5.83 (dd, J3,4 = 10.1 Hz, J3,2 = 3.4 (m, 2H, H-5b, H-4), 3.83 – 3.67 (m, 1H, H-12′), 3.66 (s, 3H, -OCH3),
Hz, 1H, H-3), 5.67 (t, J4,3 = J4,5 = 10.0 Hz, 1H, H-4), 5.60 (dd, J2,3 = 3.2, 2.42 – 2.17 (m, 4H, 2×H-11′, 2×H-2′), 2.01-2.00 (m, 2H, H-8′), 1.68 –
J
2,1 = 1.7 Hz, 1H), 5.58 – 5.44 (m, 2H, H-9′, H-10′), 5.10 (s, 1H, H-1), 1.16 (20H, -(CH2)10-), 0.86 (t, J17′, 18′ = 5.6 Hz, 3H, -CH3); 13C NMR (100
4.33 (dq, J5,CH3 = 12.4 Hz, J5,4 = 6.2 Hz, 1H, H-5), 3.76 – 3.68 (m, 1H, MHz, CDCl3) δ 174.44 (C-1′), 166.37, 165.97, 165.58 (3×C=O),
H-12′), 3.65 (s, 3H, -OCH3), 2.51 – 2.23 (m, 4H, 2×H-11′, 2×H-2′), 133.62, 133.57, 133.16, 132.66 (Ph-C), 131.88 (C-9′), 130.09, 130.00,
2.17 – 2.01 (m, 2H, 2×H-8′), 1.71 – 1.50 (m, 4H, 2×H-7′, 2×H-13′ ), 129.90, 129.34, 129.32, 128.62, 128.59, 128.44 (Ph-C), 125.83 (C-
1.45 – 1.24 (m, 19H, -(CH2)8-, -CH3), 0.89 (t, J17′,18′ = 6.8 Hz, 3H, -CH3); 10′), 104.92 (C-1), 82.55 (C-2), 80.94 (C-4), 78.07 (C-3), 77.77 (C-12′),
13C NMR (100 MHz, CDCl3) δ 174.26 (C-1′), 165.83, 165.64, 165.49 64.07 (C-5), 51.58 (-OCH3), 34.22 (C-2′), 33.79 (C-11′), 32.85 (C-13′),
(3×Ph-CO), 133.39, 133.26 133.03 (3×Ph-C), 132.25 (C-9′), 129.90, 31.93, 29.67, 29.59, 29.29, 29.24, 27.52, 25.31, 25.06, 22.75 (-
129.72, 129.68, 129.57, 129.41, 129.32, 128.56, 128.40, 128.25 (m, (CH2)10-), 14.23 (C-18′); HR MS m/z Calculated for C45H56 O11
Ph-C) 125.48 (C-10′), 96.81 (C-1), 79.46 (C-12′), 72.03 (C-4), 71.47 [M+Na]+ = 779.3371, found 779.3372.
(C-2), 70.13 (C-3), 66.78 (C-5), 51.42 (-OCH3), 34.08 (C-2′), 33.58 (C-
13′), 32.73 (C-11′), 31.79, 29.62, 29.45, 29.21, 29.14, 27.51, 25.14,
Methyl 12-O-(β-D-arabinofuranosyl)-ricinoleate (39)
The title compound was prepared from 38 (1.00 g, 1.32 mmol) by
the general procedure described above. The reaction time was 3 h;
24.95, 22.65, 22.58, 17.58 (m, -(CH2)10-), 14.11(C-18′); HR MS m/z
Calculated for C46H58 O10 [M+Na]+ = 793.3928, found 793.3933.
23
D
and 39 (540 mg) was obtained in 92% yield as a thick syrup.
= ̶
[α
]
Methyl 12-O-(α-L-rhamnopyranosyl)-ricinoleate (36)
The title compound was prepared from 35 (500 mg, 0.65 mmol) by
1
74.4° (c = 0.5, CHCl3); H NMR (400 MHz, CD3OD) δ 5.50 – 5.37 (m,
2H, H-9′, H-10′), 4.97 (d, J1,2 = 1.5 Hz, 1H, H-1), 4.00 – 3.92 (m, 2H, H-
the general procedure described above. The reaction time was 4 h;
4, H-2), 3.84 (dd, J3,2 = 6.4 Hz, J3,4 = 3.7 Hz, 1H, H-3), 3.74 (dd, J5a,5b
=
23
D
and 36 (270 mg) was obtained in 91% yield as a thick syrup.
= ̶
[α
]
11.9 Hz, J5a,4 = 3.2 Hz, 1H, H-5a), 3.69 – 3.59 (m, 5H, -OCH3, H-5b, H-
12′), 2.38 – 2.25 (m, 4H, 2×H-2′, 2×H-11′), 2.10 – 2.00 (m, 2H, 2×H-
8′), 1.66 – 1.44 (m, 4H, 2×H-7′, 2×H-13′), 1.44 – 1.25 (m, 16H, -
(CH2)8-), 0.90 (t, J17′,18′ = 6.7 Hz, 3H, -CH3); 13C NMR (100 MHz,
CD3OD) δ 176.18 (C-1′), 132.46 (C-9′), 127.00 (C-10′), 108.80 (C-1),
85.22 (C-4), 83.66 (C-2), 78.99 (C-3), 78.65 (C-12′), 62.85 (C-5), 52.05
(-OCH3), 34.81 (C-2′), 34.68 (C-13′), 34.04 (C-11′), 32.97, 30.62,
30.56, 30.22, 30.16, 30.12, 28.34, 26.26, 25.99, 23.67 (m, -(CH2)10-),
1
57.8° (c = 0.5, CHCl3); H NMR (400 MHz, CD3OD) δ 5.53 – 5.34 (m,
2H, H-9′, H-10′), 4.77 (d, J1,2 = 1.4 Hz, 1H, H-1), 3.78 (dd, J2,3 = 3.3 Hz,
J2,1 = 1.6 Hz, 1H, H-2), 3.76 – 3.67 (m, 1H, H-5), 3.67 – 3.57 (m, 5H, -
OCH3, H-3, H-12′), 3.38 (t, J4,3 = J4,5 = 9.5 Hz, 1H, H-4), 2.40 – 2.21 (m,
4H, 2×H-11′, 2×H-2′), 2.11– 2.01 (m, 2H, 2×H-8′ ), 1.67 – 1.44 (m, 4H,
2×H-7′, 2×H-13′), 1.44 – 1.26 (m, 16H, -(CH2)8-), 1.24 (d, J5,CH3 = 6.3
Hz, 3H, -CH3 ), 0.91 (t, J17′,18′ = 6.8 Hz, 3H, -CH3); 13C NMR (100 MHz,
CD3OD) δ 174.72 (C-1′), 131.42 (C-9′), 125.34 (C-10′), 99.72 (C-1),
78.02 (C-12′), 72.53 (C-4), 71.30 (C-2), 71.06 (C-3), 68.65 (C-5), 50.65
(-OCH3), 33.43 (C-2′), 33.01 (C-13′), 32.35 (C-11′), 31.59, 29.24,
29.16, 28.85, 28.81, 28.76, 26.97, 24.76, 24.62, 22.28 (m, -(CH2)10),
16.55 (-CH3), 13.06 (C-18′); HR MS m/z Calculated for C24H46 O7
[M+Na]+ = 481.3142, found 481.3154.
14.44 (C-18′); HR MS m/z Calculated for C24H44 O7 [M+Na]+
467.2985, found 467.3003.
=
12-O-(β-D-Arabinofuranosyl)-ricinoleic acid (40)
The title compound was prepared from 38 (1.00 g, 1.32 mmol) by
the general procedure described above. The reaction time was 8 h
and compound 40 (0.45 g) was obtained in 79% yield as a thick
67.0° (c = 0.5, CHCl3); 1H NMR (400 MHz, CD3OD) δ
23
D
12-O-(α-L-Rhamnopyranosyl)-ricinoleic acid (37)
The title compound was prepared from 35 (2.00 g, 2.6 mmol) by the
general procedure described above. The reaction time was 8 h; and
37 was obtained (1.00 g) in a yield of 87%) as a thick syrup.
syrup.
=
̶
[α
]
5.49 – 5.39 (m, 2H, H-9′, H-10′), 4.97 (d, J1,2 = 1.4 Hz, 1H, H-1), 4.00 –
3.94 (m, 2H, H-4, H-2), 3.84 (dd, J3,2 = 6.3 Hz, J3,4 = 3.7 Hz, 1H, H-3),
3.74 (dd, J5a,5b= 11.9 Hz, J5a,4 = 3.3 Hz, 1H, H-5a), 3.68 – 3.59 (m, 2H,
23
D
= ̶
[α
]
1
14.7° (c = 0.5, CHCl3); H NMR (400 MHz, CD3OD) δ 5.51 – 5.38 (m, H-5b, H-12′), 2.41 – 2.24 (m, 4H, 2×H-11′, 2×H-2′), 2.11 – 1.98 (m,
2H, H-9′, H-10′), 4.77 (d, J1,2 = 1.1 Hz, 1H, H-1), 3.78 (dd, J2,3 = 3.1 Hz, 2H, 2×H-8′), 1.67 – 1.45 (m, 4H, 2×H-7′, 2×H-13′), 1.45 – 1.22 (m,
J2,1 = 1.5 Hz, 1H, H-2), 3.76 – 3.69 (m, 1H, H-5), 3.68 – 3.57 (m, 2H, , 16H, -(CH2)8-), 0.90 (t, J17′,18′ = 6.7 Hz, 3H, -CH3); 13C NMR (100 MHz,
H-3, H-12′), 3.38 (t, J4,3 = J4,5 = 9.5 Hz, 1H, H-4), 2.41 – 2.20 (m, 4H, CD3OD) δ 177.89 (C-1′), 132.49 (C-9′), 126.96 (C-10′), 108.75 (C-1),
10 | J. Name., 2012, 00, 1-3
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85.19 (C-4), 83.64 (C-2), 79.01 (C-3), 78.62 (C-12′), 62.81 (C-5), 34.98 Methyl
12-O-(2,3,6,
2′,3′,4′,6′-hepta-O-acetyl-β-D-lactosyl)-
(C-2′), 34.65 (C-13′), 34.01 (C-11′), 32.95, 30.62, 30.54, 30.25, 30.18, ricinoleate (44)
30.16, 28.34, 26.24, 26.05, 23.65 (m, -(CH2)10-), 14.44 (C-18′); HR MS The title compound was prepared from acetobromolactose (20,
m/z Calculated for C23H42 O7 [M+Na]+ = 453.2829, found 453.2838.
.
5.00 g, 7.15 mmol following the general procedure described
above. The reaction time was 16 h; and 44 (3.52 g) was obtained in
15.6° (c = 0.5, CHCl3); 1H NMR
23
D
Methyl
12-O-(2,3,4,6-tetra-O-acetyl-β-D-galactofuranosyl)- 53% yield as a thick syrup.
=
̶
[α
]
ricinoleate (41)
(400 MHz, CDCl3) δ 5.44 – 5.36 (m, 1H, H-9′′), 5.36 – 5.26 (m, 2H, H-
10′′, H-4′), 5.17 (t, J3,4 = J3,2 = 9.3 Hz, 1H, H-3 ), 5.09 (dd, J2′,3′ = 10.4
Hz, J2′,1′ = 7.9 Hz, 1H, H-2′), 4.94 (dd, J3′,2′ = 10.4 Hz, J3′,4′ = 3.4 Hz, 1H,
H-3′), 4.87 (dd, J2,3 = 9.6 Hz, J2,1 = 8.0 Hz, 1H, H-2), 4.50 (d, J1,2 = 8.0
Hz, 1H, H-1), 4.47 (m, (J1′,2′ = 8.0 Hz, H-1′), 2H, H-1′, H-6′a), 4.08 (m,
The title compound was prepared from 2,3,4,6-tetra-O-acetyl-α-D-
galactofuranosyl chloride (19, 2.00 g, 5.5 mmol) by the general
procedure described above. The reaction time was 14 h; and 41
23
D
(2.12 g) was obtained in 60% yield as a thick syrup.
= ̶ 24.4° (c
[α
]
1
= 0.5, CHCl3); H NMR (400 MHz, CDCl3) δ 5.48 – 5.32 (m, 3H, H-9′, 3H, H-6a,b, H-6′b), 3.86 (m, 1H, H-5′), 3.75 (t, J4,3 = J4,5 = 9.4 Hz, 1H,
H-10′, H-5), 5.08 (s, 1H, H-1), 5.00 (dd, J2,3 = 2.0 Hz, J1,2 = 0.5 Hz, 1H, H-4), 3.65 (s, 3H, OCH3), 3.58 (m, 1H, H-5), 3.54 – 3.44 (m, 1H, H-
H-2), 4.96 (dd, J3,4 = 6.1 Hz, J3,2 = 1.7 Hz, 1H, H-3), 4.33 – 4.25 (m, 2H, 12′′), 2.36 – 2.18 (m, 4H, 2×H-2′′, 2×H-11′′), 2.15 – 1.92 (m, 23H, 7×-
H-6a, H-4), 4.18 (dd, J6a,6b = 11.7 Hz, J6b,5 = 7.3 Hz, 1H, H-6b), 3.65 (s, OCOCH3, 2×H-8′′), 1.59 (m, 2H, 2×H-7′′), 1.38 (m, 2H, 2×H-13′′), 1.33
3H, -OCH3), 3.63 – 3.53 (m, 1H, H-12′), 2.32 – 2.18 (m, 4H, 2×H-2′, – 1.15 (m, 16H, -(CH2)8-), 0.86 (t, J18′′,17′′= 6.9 Hz, 3H, -CH3); 13C NMR
2×H-11′), 2.12, 2.09, 2.06, 2.03 (4×s, 12H, 4×OCOCH3), 2.03 – 1.92 (100 MHz, CDCl3) δ 174.30 (C-1′′), 170.37, 170.34, 170.17, 170.08,
(m, 2H, 2×H-8′), 1.66 – 1.54 (m, 2H, 2×H-7′), 1.52 – 1.40 (m, 2H, 169.89, 169.52, 169.11 (7×OCOMe), 131.86 (C-9′′), 124.98 (C-10′′),
2×H-13′), 1.39 – 1.17 (m, 16H, -(CH2)8-), 0.86 (t, J17′,18′ = 6.8 Hz, 3H, - 101.09 (C-1), 100.85 (C-1′), 81.89, (C-12′′) 76.56 (C-4), 73.03 (C-3),
CH3); 13C NMR (100 MHz, CDCl3) δ 174.38 (C-1′), 170.61, 170.17, 72.44 (C-5), 72.01 (C-2), 70.99 (C-3′), 70.66 (C-5′), 69.11 (C-2′), 66.62
170.16, 169.79 (4×OCOCH3), 131.96 (C-9′), 125.70 (C-10′), 104.87 (C- (C-4′), 62.15 (C-6′), 60.82 (C-6), 51.46 (COOCH3), 34.08 (C-13′′),
1), 81.89 (C-2), 79.73 (C-4), 78.21 (C-12′), 76.64 (C-3), 69.33 (C-5), 33.94 (C-8′′), 33.18 (C-11′′), 31.86, 29.50, 29.39, 29.18, 29.14, 29.11,
62.85 (C-6), 51.56 (-OCH3), 34.20 (C-2′), 33.74 (C-13′), 32.87 (C-11′), 27.42, 25.19, 24.93, 22.63 (m, -(CH2)8-), 20.83, 20.81, 20.70, 20.64,
31.90, 29.67, 29.49, 29.30, 29.29, 29.23, 27.54, 25.23, 25.05, 22.72, 20.52 (m, 7×OCOCH3), 14.07 (C-18′′); HR MS m/z Calculated for
20.94, 20.92, 20.81 (m, -(CH2)10-), 14.20 (C-18′); HR MS m/z C45H70 O20 [M+Na]+ = 953.4358, found 953.4364.
Calculated for C33H54 O12 [M+Na]+ = 665.3513, found 665.3547.
Methyl 12-O-(β-D-lactosyl)-ricinoleate (45)
Methyl 12-O-(β-D-galactofuranosyl)-ricinoleate (42)
The title compound was prepared from 44 (500 mg, 0.54 mmol)
The title compound was prepared from 41 (250 mg, 0.38 mmol) following the general procedure described above. The reaction time
following the general procedure described above. The reaction time was 1 h; and 45 (310 mg) was obtained in 91% as a thick syrup.
1
23
D
was 0.5 h; and 42 (170 mg) was obtained in 94% yield as a thick
= +68.4° (c = 0.5, MeOH); H NMR (400 MHz, CD3OD) δ 5.51 –
[α
]
syrup.
= ̶
36.6° (c = 0.5, CHCl3); 1H NMR (400 MHz, CD3OD) δ
23
[α
]
5.37 (m, 2H, H-9′′, H-10′′), 4.38 (d, J1,2= 7.4 Hz, 1H, H-1), 4.37 (d, J1′,2′
D
5.49 – 5.39 (m, 2H, H-9′, H-10′), 4.95 (d, J1,2 = 1.5 Hz, 1H, H-1), 4.00 = 7.8 Hz, 1H, H-1′) 3.89 – 3.36 (m,15H, H-3′, H-4′, H-5′, H-6′a,b, H-2,
(dd, J3,4 = 6.5 Hz, J4,5 = 4.1 Hz, 1H, H-4), 3.95 –3.93 (m, 2H, H-2, H-3), H-3, H-4, H-5, H-6a,b, H-12′′, -OCH3), 3.24 (dd, J2′,3′ = 8.9Hz, J2′,1′ = 8.0
3.72 (m, 1H, H-5), 3.64 – 3.56 (m, 6H, -OCH3, H-12′, H-6a,b), 2.41 – Hz, 1H, H-2′), 2.32 (m, 4H, 2×H-2′′, 2×H-11′′), 2.06 (m, 2H, H-8′′ ),
2.23 (m, 4H, 2×H-2′, 2×H-11′ ), 2.12 – 1.97 (m, 2H, 2×H-8′), 1.67 – 1.63 – 1.56 (m, 2H, 2xH-7′′), 1.56 – 1.47 (m, 2H, 2xH-13′′), 1.42 –
1.52 (m, 2H, 2×H-7′), 1.55 – 1.42 (m, 2H, 2×H-13′), 1.42 – 1.22 (m, 1.20 (m, 16H, -(CH2)8-), 0.90 (t, J18′′,17′′= 6.8 Hz, 3H, -CH3); 13C NMR
16H, -(CH2)8-), 0.90 (t, J17′,18′ = 6.7 Hz, 3H, -CH3); 13C NMR (100 MHz, (100 MHz, CD3OD) δ 176.12 (C-1′′), 132.47 (C-9′′), 126.86 (C-10′′),
CD3OD) δ 176.13 ( C-1′), 132.45 (C-9′), 127.06 (C-10′), 108.90 (C-1), 105.09 (C-1), 104.16 (C-1′), 81.43, 80.78, 77.11, 76.49, 76.33, 74.94,
84.11 (C-2), 83.62 (C-3) 79.26 (C-6), 78.63 (C-4), 72.25 (C-5), 64.89 74.78, 72.60, 70.32, 62.51, 62.09 (m), 52.07 (-OCH3), 34.84, 34.23,
(C-12′), 52.03 (OCH3), 34.81 (C-2′), 34.07 (C-13′), 32.98 (C-11′), 33.05, 30.71, 30.68, 30.32, 30.3, 30.20, 28.45 26.15, 26.05, 23.75
30.65, 30.57, 30.25, 30.18, 30.14, 28.37, 26.29, 26.00, 23.68(m, - (m, -(CH2)8-), 14.52 (C-18′′); HR MS m/z Calculated for C31H56O13
(CH2)10-), 14.45 (C-18′); HR MS m/z Calculated for C25H46 O8 [M+Na]+ [M+Na]+ = 659.3619, found 659.3622.
= 497.3091, found 497.3111.
12-O-(β-D-Lactosyl)-ricinoleic acid (46)
12-O-(β-D-Galactofuranosyl) ricinoleic acid (43)
The title compound was prepared from 44 (200 mg, 0.21 mmol)
The title compound was prepared from 41 (300 mg, 0.46 mmol) following the general procedure described above. The reaction time
following the general procedure described above. The reaction time was 4 h; and 46 (110 mg) was obtained in 84% yield as a thick syrup.
1
23
D
was 3 h; and 43 (181 mg was obtained in 85% yield as a thick syrup.
= +39.2° (c = 0.5, MeOH); H NMR (400 MHz, CD3OD) δ 5.49 –
[α
]
= ̶
31.2° (c = 0.5, CHCl3); 1H NMR (400 MHz, DMSO) δ 5.37 (m,
23
[α
]
5.41 (m, 2H, H-9′′, H-10′′ ) 4.41 (d, J1,2 = 7.0 Hz, 1H, H-1), 4.39 (d, J1
D
′,2′
2H, H-9′, H-10′), 4.74 (s, 1H, H-1), 3.91 – 3.24 (m, 7H, H-4, H-2, H-3, = 7.7 Hz, 1H, H-1′), 3.92 – 3.77 (m, 4H, H-6a′, H-6b′, H-4′, H-6a), 3.76
H-5, H-12′ H-6a,b), 2.34 – 2.07 (m, 2H, H-11′), 2.05 – 1.84 (m, 4H, – 3.67 (m, 2H, H-6b, H-12′′), 3.66 – 3.49 (m, 5H, H-4, H-5, H-3′, H-2,
2×H-2′, 2×H-8′), 1.58 – 1.09 (m, 20H, -(CH2)10-), 0.84 (t, J18′,17′ = 6.6 H-3), 3.47 – 3.39 (m, 1H, H-5′), 3.27 (t, J2′,3′ = J2′,1′ = 8.0 Hz, 1H, H-2′),
Hz, 3H, CH3 ); 13C NMR (100 MHz, DMSO) δ 174.46 (C-1′), 131.07 (C- 2.50 – 2.19 (m, 4H, 2×H-11′′, 2×H-2′′), 2.13 – 2.00 (m, 2H, H-8′′),
9′), 126.18 (C-10′), 107.56 (C-1), 82.56 (C-2), 82.06 (C-3), 77.00 (C- 1.68 – 1.43 (m, 4H, 2×H-7′′, 2×H-13′′), 1.44 – 1.23 (m, 16H, -(CH2)8-),
12′), 76.74 (C-4), 70.48 (C-5), 63.36 (C-6), 36.84 (C-2′), 33.53 (C-13′), 0.91 (t, J18′′,17′′ =6.8 Hz, 3H, -CH3); 13C NMR (100 MHz, CD3OD) δ
32.89 (C-11′), 31.43, 29.23, 29.03, 28.82, 27.01, 25.82, 24.80, 22.23 173.99 (C-1′′), 131.23 (C-9′′), 125.28 (C-10′′), 103.61 (C-1), 102.70
(m, -(CH2)10-) 14.12 (C-18′); HR MS m/z Calculated for C24H44 O8 (C-1′), 80.15 (C-12′), 79.28 (C-4), 75.65 (C-5), 75.06 (C-2′), 74.90 (C-
[M+Na]+ = 483.2934, found 483.2933.
5′), 73.51 (C-2), 73.36 (C-3), 71.20 (C-3′), 68.89 (C-4′), 61.09 (C-6),
60.62 (C-6′), 36.05 (C-2′′), 33.37 (C-13′′), 32.76 (C-11′′), 31.58, 29.34,
29.20, 29.06, 28.96, 27.07, 25.66, 24.70, 22.30 (m, -(CH2)8-), 13.10
This journal is © The Royal Society of Chemistry 20xx
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(C-18′′); HR MS m/z Calculated for C30H54O13 [M+Na]+ = 645.3462, stirring for a few minutes the reaction mixture was diluted with
found 645.3470.
diethyl ether and was washed successively with water (x3) and
brine in a separatory funnel in the cold. The ether layer was then
Methyl
9,10-dibromo-12-O-(2,3,4,6-tetra-O-acetyl-β-D- dried over anhydrous Na2SO4 and was concentrated to dryness
glucopyranosyl)-ricinoleate (47)
under reduced pressure. Purification of the product by
Bromine (0.05 mL, 0.87 mmol) was added to a stirred solution of 24 chromatography on silica (eluent, EtOAc: hexane) yielded 49 and
1
(370 mg, 0.58 mmol) in chloroform (5 mL) at room temperature and 49a (140 mg; 1: 0.5 by H NMR) as a mixture. The mixture (30 mg)
the stirring was continued for 5 h at which time TLC showed was therefore dissolved in anhydrous MeOH and was treated with
complete consumption of 24. The reaction mixture was diluted with NaOMe (catalytic) for 2 h at room temperature. Amberlite IR 120 H+
CH2Cl2 (100 mL) and was washed successively water, aqueous resin was then added to it to acidic pH and the solution on
NaHCO3, and brine solutions in the cold. The organic layer was then concentration to dryness and filtration through a short column of
23
D
dried over anhydrous Na2SO4 and was concentrated under reduced silica afforded the title compound neat (20 mg, 72 %).
= +19.3°
[α
]
1
pressure to yield, after chromatography on a column of silica gel
(c = 0.3, CHCl3); H NMR (400 MHz, CDCl3) δ 7.37-7.22 (m, 5H, Ph-
CH), 5.48-5.35 (m, 2H, H-9, H-10), 4.57 (d, JA,B= 11.7 Hz, 1H, Ph-
CHH), ), 4.49 (d, JA,B = 11.7 Hz, 1H, Ph-CHH), 3.66 (s, 3H, -COOCH3),
(eluent, EtOAc: hexane), the title compound 47 (370 mg, 80%) (as a
23
D
pair of enantiomers) in the form of a thick syrup.
= + 41.6° (c =
[α
]
0.25, CHCl3); 1H NMR (400 MHz, CDCl3) (The two enantiomers are 3.40 (p, J = 6.0 Hz, 1H, H-12), 2.36-2.23 (m, 4H, 2×H-2, 2×H-11), 2.03
marked A & B) δ 5.45 (dd, 2H, J3,4 = 9.5 Hz, J4,3 = 9.9 Hz H-3A, H-3B), (q, 2H, J= 6.4 Hz, J= 6.7 Hz, 2×H-8), 1.65-1.22 (m, 20 H -(CH2)10), 0.88
5.17-5.13 (2×d, J1,2 = 5.1 Hz, 2H, H-1A, H-1B), 5.06 (2×t, J4,5 = J4,3
=
(t, J18,17 =6.8 Hz, 3H, -CH3); 13C NMR (100 MHz, CDCl3) δ 174.43 (C-
10.0 Hz, 2H, H-4A, H-4B), 4.97-4.87 (2×d, J2,3 = 10.4 Hz, J2,1 = 3.7 Hz, 1), 139.20 (Ph-C), 131.85 (C-9), 128.40, 127.86, 127.52 (Ph-C),
2H, H-2A, H-2B), 4.29-4.03 ( m, 10H, 2×H-6a, 2×H-5, 2×H-9′, 2×H-10′, 125.69 (C-10), 79.14 (C-12), 71.07 (Ph-CH2), 51.57 (-OCH3), 34.25 (C-
2×H-6b), 3.85 -3.76 (m, 2×H-12′), 3.66 (s, 6H, 2×-OCH3), 2.30 (t, 4H, 2), 34.13 (C-13), 32.02 (C-11), 31.80, 29.71, 29.59, 29.32, 29.29,
J
2′,3′ = 7.5 Hz, 4×H-2′), 2.1-2.0 (7×s, 24H, 8×OCOCH3), 1.67-1.57 (m, 29.27, 27.57, 25.59, 25.10, 22.79 (m, -(CH2)9-), 14.09 (C-18); HR MS
48H, 2× -(CH2)12), 0.88 (t, J18′,17′ = 6.8 Hz, 6H, 2×CH3); 13C NMR (100 m/z Calculated for C26H42 O3 [M+Na]+ = 425.3032, found 425.3035.
MHz, CDCl3) δ 174.38 (2×C-1′), 170.80, 170.35, 170.29, 170.18,
170.10, 169.73, 169.71 (m, 8×OCOCH3), 98.23 (C-1A), 95.17 (C-1B), Methyl 12-O-methyl-ricinoleate (50)
81.01 (C-12′A), 78.38 (C-12′B), 70.98 (C-2), 70.25 (C-3), 68.80 (C-4A), The title compound was prepared from 7 (313 mg, 1 mmol) using
68.63 (C-4B), 67.94 (C-5), 62.21 (C-6A), 62.08 (C-6B), 59.86 (C-9′A), NaH(70 mg, 1.5 mmol) and MeI (0.09 mL, 1.5 mmol) in DMF (5.0
59.14 (C-9′B), 56.69 (C-10′A), 54.32 (C-10′B), 51.61 (-OCH3), 40.53, mL) by the same procedure as described for 49. The reaction time
1
35.67, 35.37, 34.04, 33.70, 31.75, 29.50, 29.24, 29.00, 28.66, 28.63, was 4 h and product obtained (210 mg) was a mixture (1:0.5 by H
27.69, 25.08, 24.87, 22.62, 22.56, 21.00, 20.82, 20.71, 20.63 (m, NMR) of the ester 50 and the lactone 50a. Treatment of the mixture
2×(CH2)13), 14.21 (C-18′A); 14.17 (C-18′B); HR MS m/z Calculated for (60 mg) with a solution of anhydrous HCl in MeOH (prepared by
C35H54Br2O12 [M+Na]+
[M+2+Na]+.
=
823.1880, found 823.1880, 825.1863 reacting 0.02 mL thionyl chloride with anhydrous MeOH, 3 mL, at -5
°C) under the nitrogen atmosphere for 8 h at room temperature,
followed by concentration of the reaction mixture to dryness and
Methyl 9,10-dibromo-12-O-(β-D-glucopyranosyl)-ricinoleate (48)
purification of the residue by filtration through a column of silica
The title compound was prepared from 47 (45 mg, 0.06 mmol) (eluent, EtOAc: hexanes) gave 50 as a neat product (32 mg, 80%
= +17.2° (c = 0.25, CHCl3); 1H NMR (400
23
D
following the general procedure described above. The reaction time based on 50 weight).
[α
]
23
was 0.5 h; and 48 (31 mg, 87%) was obtained as a thick syrup.
[α
]
MHz, CDCl3) δ 5.46-5.33 (m, 2H, H-9, H-10) 3.65 (s, 3H, -COOCH3),
D
= +37.1° (c = 0.75, CHCl3); 1H NMR (400 MHz, CD3OD) (The two 3.32 (s, 3H, -OCH3), 3.17 (p, J= 5.8 Hz, 1H, H-12), 2.31-2.15 (m, 4H, ,
enantiomers are marked A & B) δ, 4.89- 4.78 (2×d, J1,2 = 4.0 Hz, 2H, 2×H-2, 2×H-11), 2.01 (q, 2H, J= 6.5 Hz, J= 6.7 Hz, 2×H-8), 1.65 – 1.53
H-1A, H-1B), 4.39-4.35 (m, 2H, H-4A, H-4B), 4.26-4.22 (m, 2H, H-3A, (m, 2H, 2×H-7), 1.48 – 1.37 (m, 2H, 2×H-13),1.37-1.21 (m, 16 H -
H-3B), 4.14-4.10 (m, 2H, H-5A, H-5B), ), 3.81-3.48 ( m, 16H, 2×H-12′, (CH2)8-), 0.86 (t, J18,17 =6.6 Hz, 3H, -CH3); 13C NMR (100 MHz, CDCl3) δ
2×H-6a, 2×H-6b, 2×-OCH3, 2×H-9′, 2×H-10′), 3.30-3.24 (m, 2H, H-2A, 174.23 (C-1), 131.67 (C-9) 125.42 (C-10), 80.97 (C-12), 56.54 (-
H-2B), 2.24-1.22 (m, 52H, 2× -(CH2)13), 0.88 (t, J18′,17′ = 6.9 Hz, 6H, OCH3), 51.39 (-COOCH3), 34.07 (C-2), 33.57 (C-13), 31.86 (C-11),
2×CH3); 13C NMR (100 MHz, CD3OD) δ 174.66 (2×C-1′), 101.14 (C- 31.86, 31.06, 29.54, 29.47, 29.11, 27.38, 25.34, 24.93, 22.61, (m, -
1A), 98.61 (C-1B), 78.72 (C-12′A), 76.99 (C-12′B), 73.59, 73.52, (CH2)9-), 14.06 (C-18); HR MS m/z Calculated for C20H38O3 [M+Na]+ =
72.69, 72.47, 72.42, 72.13 (m, 2×C-6, 2×C-9′, 2×C-10′), 70.36 (C-2A), 349.2719, found 349.2714.
70.22 (C-2B), 61.15, 61.03, 60.92, 59.58, 57.42, 55.43, 53.32 (m,
Conclusions
2×C-5, 2×C-3, 2×C-4) 50.61 (2×OCH3), 43.26, 40.88, 37.34, 36.55,
In conclusion, 28 novel glycosides of ricinoleic acid were
35.53, 34.23, 33.44, 31.54, 29.15, 29.01, 28.71, 28.68, 28.65, 28.41,
synthesized and their antimicrobial activity was evaluated, seven of
28.39, 27.18, 27.07, 24.84, 24.71, 24.56, 22.30, 22.24 (m, 2×(CH2)13),
them showing promising wide spectrum antibacterial activity
13.01 (C-18′A); 12.99 (C-18′B); HRMS m/z Calculated for C25H46Br2
O8 [M+Na]+ = 655.1457, found 657.1448 [M+2+Na]+.
against Gram +ve bacteria. Two compounds, 29 (NP-2672) and 39
(NP-2689), showed good to excellent activity against various non-
clinical/clinical/NorA overexpressed/resistant strains of S. aureus as
Methyl 12-O-benzyl-ricinoleate (49)
well as other Gram +ve bacteria. These results suggest that
NaH (46 mg, 0.96 mmol) was added to a solution of methyl
carbohydrate moiety in the glycosides of the ricinoleic acid played
ricinoleate (7, 200 mg, 0.6 mmol) in DMF (5 mL) at 0 °C and the
an important role for imparting the antibacterial activity by
mixture was stirred for 15 min. BnBr (0.1 mL, 0.76 mmol) was then
increasing the membrane permeability. It is presumably because of
added to the mixture and the temperature was allowed to be
binding of these molecules with D-Ala-D-Ala, preventing the
brought to 10 °C, and the stirring was continued for 10 h. At this
synthesis of the long polymers of N-acetylmuramic acid and N-
time TLC showed complete disappearance of 7. MeOH (1 mL)
acetylglucosamine that form the backbone strands of the bacterial
followed by triethyl amine (1 mL) were added to the mixture. After
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cell wall. In this sense, these compounds could be potential 17
candidates for therapeutics as they may not be targeting the cell
components such as nucleic acids and proteins. The future studies
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K. R. K. sincerely acknowledges financial support in the form of a
Senior Research Fellowship by CSIR, India. H. N. and R. T. sincerely
acknowledge financial support from CSIR-Network Project (BSC-
211) to carry out the work and the Department of Biotechnology,
Govt. of India for Junior Research Fellowship, respectively. This
work is dedicated to Chandravathi Kuppala and Gangaraju Kuppala.
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Carbohydr. Res., 2010, 2, 20–27.
TABLE OF CONTENTS GRAPHIC
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39
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Synthesis and antibacterial activity of ricinoleic acid
glycosides
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Ramakrishna Kuppala, Mugunthan Govindarajan., Rushikesh
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K. P. Ravindranathan Kartha
O
40
G. J. F. Chittenden, Carbohydr. Res., 1972, 25, 35–41.
O
MeO
R2
O
O
HO
O
HO
O
O
AUTHOR INFORMATION
Corresponding Author
MIC = 2 µg/ml
S. aureus 1199
R1O
OH
a *Department of Medicinal Chemistry, National Institute of Pharmaceutical
Education and Research, S.A.S. Nagar, Punjab-160062, India.
*Email: rkartha@niper.ac.in; Fax: +91-(0)-172-2214692.; Tel: +91-(0)-172-
2292028
R1O
O
MIC = 4 µg/ml
S. aureus 1199B
OH
HO
O
R2
HO
HO
O
O
Co-corresponding Author
OMe
R1 = H, Ac, Bz; R2 = OH, OMe, O-Na+, O-Li+
b**Bioactive Screening Laboratory, CSIR-Institute of Microbial Technology,
Sector-39A, Chandigarh-160036, India
O
**Email: hemraj@imtech.res.in; Fax: + 91-(0)-172-2690585; Tel: +91-(0)-
172-6665338
c Department of Natural Products, National Institute of Pharmaceutical
Education and Research, S.A.S. Nagar, Punjab-160062, India.
Author Contributions
All authors have given approval to the final version of the manuscript.
Electronic Supplementary Information (ESI) available:www.rsc.org/
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Figure 1: Ricinoleic acid-derived compounds known in the literature as anti-bacterial and antimycobacterial agents.
Reagents and conditions: (i) HCl/MeOH, rt, 4 h, 90%; (ii) Ag2CO3, AgClO4, powdered molecular sieves (4Å), CH2Cl2 rt, 10-12 h, 53-68%; (iii)
NaOMe/MeOH, 20-40 min, rt, 90-94%; (iv) LiOH/aq THF, 2-4 h, rt, 80-87%; (v) Amberlite IR 120 (H+), rt, 80-88%
Scheme1. Synthesis of ricinoleic acid glycosides in their partially/fully deprotected form and/as their sodium/lithium salts.
Table 1. Different ricinoleic acid glycosides and their partially or fully deprotected and sodium/lithium salt derivatives
Acetohalosugar
Glycosylated ricinoleates synthesized
Structure Compound number
Yield (%)
used for synthesis
21, R1 = Ac, R2 = OCH3
22, R1 = H, R2 = OCH3
68
92
23, R1 = H, R2 = OH
87
58
13
14
25
24, R1 = Ac, R2 = OCH3
25, R1 = H, R2 = OCH3
26, R1 = H, R2 = OH
90
81
27, R1 = H, R2 = O-Li+
28, R1 = Ac, R2 = OCH3
83
60
29, R1 = H, R2 = OCH3
30, R1 = H, R2 = OH
94
85
15
31, R1 = H, R2 = O-Na+
32, R1 = Ac, R2 = OCH3
80
52
33, R1 = H, R2 = OCH3
34, R1 = H, R2 = OH
92
83
16

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35, R1 = Bz, R2 = OCH3
36, R1 = H, R2 = OCH3
66
91
17
18
19
37, R1 = H, R2 = OH
87
57
38, R1 = Bz, R2 = OCH3
39, R1 = H, R2 = OCH3
92
40, R1 = H, R2 = OH
79
60
41, R1 = Ac, R2 = OCH3
42, R1 = H, R2 = OCH3
94
43, R1 = H, R2 = OH
85
53
44, R1 = Ac, R2 = OCH3
45, R1 = H, R2 = OCH3
91
20
24
46, R1 = H, R2 = OH
84
80
47, R1 = Ac, R2 = OCH3
(For the structure,
see the entry at row
2 above)
48, R1 = H, R2 = OCH3
87
0.6
0.5
0.4
0.3
0.2
0.1
0
Vancomycin
29
39
Control
Time (H:MM:SS)
Figure 2: The cytoplasmic membrane permeabilization of S. aureus cells treated with compounds 29 (squares), 39 (triangles) and
vancomycin (diamonds).The untreated S. aureus cells (crosses) were taken as control. (Compounds 29 and 39 induced an increase in the
permeability of S. aureus. The experiment was carried out two times in triplicate. The results are presented as mean ± SD.)

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900
800
700
600
500
400
300
200
100
0
Vancomycin
29
39
control
Time (in minutes)
Figure 3: Effect of 29 (squares), 39 (triangles) and vancomycin (diamonds) on membrane integrity of S. aureus cells by propidium iodide
uptake assay. The untreated S. aureus cells (crosses) were taken as control. For each sample 106 CFU ml-1 were analyzed. The membrane
integrity of S. aureus cells was destroyed by 29 and 39. The experiment was carried out two times in triplicate. The results were presented as
mean ± SD
.
Figure 4: Cytotoxicity study of biologically active glycosides of methyl ricinoleate (Results are expressed as mean ± SD of three
independent experiments performed in triplicates)