Comparative Antibacterial Activities of Some Monosaccharide and Disaccharide Benzoates

For comparative antibacterial studies a number of furanose (3, 5), pyranose (7, 9, 11, 13), and disaccharide benzoates (14, 15) were prepared by direct benzoylation method. Synthesized benzoates (3, 5, 7, 9, 11, 13-15) along with some starting materials were screened for in vitro antibacterial activity against ten human pathogenic bacteria viz. Bacillus subtilis, Bacillus cereus, Bacillus megaterium, Staphylococcus aureus, Escherichia coli, INABA ET (Vibrio), Pseudomonas species, Salmonella paratyphi, Salmonella typhi, and Shigella dysenteriae. The study revealed that the pyranose benzoate derivatives (7, 9, 11, 13) were more prone towards antibacterial functionality than that of the furanose benzoate (3, 5) and disaccharide benzoates (14, 15).


INTRODUCTION
Monosaccharides are wide spread in nature, being a component of some plant glycosides and bacterial polysaccharides of immunological importance [1,2].In monosaccharides, protection of a particular hydroxyl group is not only necessary for the modification of the remaining functional groups but also for the synthesis of newer derivatives of great importance [3,4].The most common hydroxylprotecting acyl groups are acetyl, benzoyl etc.Both the acetyl and benzoyl groups are very cheap protecting groups that can be easily removed and the parent alcoholic components can be recovered under basic or acidic conditions [5].Per-O-acetyl and per-O-benzoyl derivatives of sugars are important intermediates in carbohydrate transformation and synthesis [6].Acetylation and benzoylation of carbohydrates will convert unprotected and polar sugars into substances soluble in many organic solvents and the resulting sugar peracetates and perbenzoates have been utilized as glycosyl donors in monosaccharide transformations and oligosaccharide syntheses [7].In comparison with sugar peracetates, sugar perbenzoates even possess some additional advantages.For example, benzoylated sugar derivatives are significantly less reactive than their acetylated analogues and tend to be more bench-stable [6,8].
Monosaccharides and nucleosides in combination with acyl nuclei (e.g.acetyl, mesyl, benzoyl, etc) play an important role as common denominator for various biological activities, which is also revealed by a number of our previous works [9][10][11].Catelani et al. [12] reported the synthesis of various 3-O-acyl-1,2-O-isopropylidene-Dglucofuranose derivatives (1a-c) from triol 4 (Figure 1) and tested their effects in augmenting the proportion of benzidine-positive (hemoglobincontaining) cells in treated K562cell populations.The results obtained demonstrated that two of these newly synthesized compounds (1b and 1c) were potent inducers of erythroid differentiation of K562cells.Very recently, we reported the synthesis and comparative antimicrobial studies of a number of furanose, pyranose and disaccharide acetates [13], and acylates [14].The structure activity relationship

Full Paper
Orbital: Electron.J. Chem.7 (2): 160-167, 2015 161 (SAR) study revealed that the pyranose acetate derivatives were more prone towards antimicrobial functionality than those of the furanose and disaccharide acetates.Considering the synthetic and biological importance, we were interested to extend our research work for the synthesis of some benzoyl derivatives of various monosaccharides and disaccharides for antibacterial studies.

General experimental procedures
Evaporations were performed under diminished pressure on a Büchi rotary evaporator.Melting points (mp) were determined on an Electrothermal melting point apparatus and are uncorrected.FT-IR spectra were recorded on a FT IR spectrophotometer (Shimadzu, IR Prestige-21) using KBr and CHCl3 technique.Thin layer chromatography was performed on Kieselgel GF254 and visualization was accomplished by spraying the plates with 1% H2SO4 followed by heating the plates at 150-200 ºC until coloration took place.Column chromatography was carried out with silica gel (100-200 mesh). 1 H (400 MHz) and 13 C (100 MHz) NMR spectra were recorded using CDCl3 as a solvent.Chemical shifts were reported in unit (ppm) with reference to TMS as an internal standard and J values are given in Hz.All reagents used were commercially available (Aldrich) and were used as received unless otherwise specified.

General procedure for direct benzoylation:
To a solution of the hydroxyl compound in anhydrous pyridine (1 mL) was added benzoyl chloride at 0 ºC followed by addition of catalytic amount of 4dimethylaminopyridine (DMAP).The reaction mixture was allowed to attain room temperature and stirring was continued for 10-16 h.A few pieces of ice was added to the reaction mixture to decompose unreacted (excess) benzoyl chloride and the reaction mixture was extracted with dichloromethane (DCM, 35 mL).The organic (DCM) layer was washed successively with 5% hydrochloric acid, saturated aqueous sodium hydrogen carbonate solution, and brine.The DCM layer was dried and concentrated under reduced pressure.The residue thus obtained on column chromatography (n-hexane/ethyl acetate) gave the corresponding benzoyl product.

Antibacterial screening tests
In vitro antibacterial functionality was evaluated against ten bacterial pathogens.Of these four were Gram-positive viz.For the detection of antibacterial activities, the disc diffusion method described by Bauer et al. [19] was followed.Dimethylformamide (DMF) was used as a solvent to initially prepare desired solution (1%) of the compounds.The plates were incubated at 37 °C for 48 h.Proper control was maintained with DMF without chemicals.Mueller-Hinton (agar and broth) medium was used for culture of bacteria.All the results were compared with the standard antibacterial antibiotic ampicillin (50 μg/disc, Beximco Pharmaceuticals Ltd., Bangladesh).Each experiment was carried out in triplicate.

Synthesis of pyranose benzoates
For pyranose sugar benzoates, our first effort was to prepare methyl tetra-O-benzoyl-Dglucopyranoside.Thus, benzoylation of methyl -Dglucopyranoside (6) afforded a white solid, mp 90-92 °C in 85% yield.The FTIR spectrum of this solid exhibited no band for hydroxyl stretching.It also showed bands at 1746, 1732 and 1714 cm 1 corresponding to carbonyl frequency and thus indicated the attachment of benzoyloxy groups in the molecule.In the 1 H NMR spectrum, a three-proton singlet at  3.50 was due to glycosidic (C-1) methoxy group.Also, H-1 appeared at  5.27 as doublet with small coupling constant (J = 3.6 Hz) indicating the glucosidic nature.In addition, the appearance of twenty protons in the aromatic region were assigned for the four benzoyloxy groups.Thus the structure was accorded as methyl 2,3,4,6-tetra-O-benzoyl--Dglucopyranoside (7).
Perbenzoylation of lactose, having eight hydroxyl groups, was conducted with the treatment with excess benzoyl chloride in anhydrous pyridine and afforded a semi-solid in 69% yield.FT-IR spectrum of the compound showed signals at 1733 and 1717 cm 1 corresponding to the carbonyl stretching peaks.Also, absence of frequency corresponding to hydroxyl group indicated the per-Obenzoylation of the molecule.In the 1 H NMR spectrum, two broad multiplets at  7.88-8.19(14H, Ar-H) and 7.15-7.76(26H, Ar-H) totaling forty aromatic protons indicated the attachment of eight benzoyloxy groups.The appearance of H-1 at  6.75 as doublet with small coupling constant (J = 3.6 Hz) indicated that it is equatorially oriented.So, the glycosidic (C-1) benzoyl group must be .Therefore, the structure was assigned as 1′,2′,3′,6′,2,3,4,6-octa-Obenzoyl--lactose (15).

Structure activity relationship (SAR)
Incorporation of benzoyl group increased the antimicrobial potentiality of different manosaccharide and disaccharides (Table 1 and Table 2).These synthesized benzoates were more active against Gram-negative pathogens than that of Gram-positive bacterial organisms.Benzoylated sugars with fivemembered furanose form are less effective against both Gram-negative and Gram-positive than that of the corresponding six-membered pyranose form.This is because of the slight distortion of furanose ring in the presence of 1,2-O-isopropylidene ring.But monosaccharides (6-13) in pyranose form with regular 4 C1 or 1 C4 conformation exhibited better antibacterial potentiality.An important observation was that, compounds 2, 4, 6, and 12 showed poor toxicity (except mannopyranoside 10) than that of partially or fully benzoylated compounds 5, 9, 11, and 13 against the tested pathogens.This is probably due to the presence of more hydroxyl groups in 2, 4, 6, and 12.While compounds 5, 9, 11, and 13 having fewer or no hydroxyl groups showed much better antimicrobial potentiality.The hydrophobicity of the molecules increased gradually from compound 2, 4, and 6 and 12 to 5, 9, 11, and 13, which is an important parameter with respect to such bioactivity and toxicity or alteration of membrane integrity, because it is directly related to membrane permeation [20].A similar hydrophobic interaction might occur between the benzoyl groups of glycopyranoses accumulated in the lipid like nature of the bacterial membranes.As a consequence of their hydrophobic interaction, bacteria lose their membrane permeability, ultimately causing death of the organisms [20][21][22].
In vitro antimicrobial activities of similar type of monosaccharide and disaccharide acetates [13] and structure activity relationship study also exhibited the almost similar inhibitory properties.Although, we expected better antibacterial potentiality for the benzoates because a large number of biologically active compounds contain aromatic and heteroaromatic nuclei [23][24][25].An important observation between these two series was that both acetates and benzoates are more prone against Gram-

CONCLUSION
Thus, we have successfully synthesized benzoylated furanose (3,5), pyranose (7, 9, 11, 13) and disaccharide (14,15) derivatives.A comparative study of in vitro antibacterial activities of monosaccharide (furanose and pyranose forms) benzoates with disaccharide benzoates was also carried out.The structure activity relationship (SAR) study revealed that the benzoylated sugar derivatives were more prone towards Gram-negative organisms than that of Gram-positive organisms.Benzoylated monosaccharides were strong inhibitors towards antibacterial functionality than that of the corresponding disaccharides.