Comparative Antimicrobial Activities of some Monosaccharide and Disaccharide Acetates

A number of furanose (2,4) and pyranose (5,7,9,11,13) acetates were prepared by direct acetylation method. For comparative antimicrobial studies sucrose octaacetate (14) was also prepared. All the compounds (1-14) 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. These compounds were also screened for in vitro antifungal activity against four pathogenic fungi viz. Aspergillus niger, Alternaria alternata, Curvularia lunata and Fusarium equiseti. The study revealed that the pyranose acetate derivatives (5,7,9,11,13) are more prone towards antimicrobial functionality than those of the furanose acetates (2,4) and sucrose octaacetate (14).


Introduction
Acyl and alkyl glycoses and glycoside derivatives of carbohydrates have immense importance and some of them have potential biological activities [1].Protection of a particular functional group of carbohydrates, especially monosaccharides, is not only necessary for the modification of the remaining functional groups but also for the synthesis of newer derivatives of great importance [2].Various methods for acylation of carbohydrates and nucleosides have so far been developed and employed successfully [3][4][5][6].A large number of biologically active compounds possess aromatic and heteroaromatic nuclei.It is also known that if an active nucleus is linked to another nucleus, the resulting nucleus may possesses greater potential for biological activity [7].The benzene and substituted benzene nuclei play important role as common denominator for various biological activities, which is also revealed in a number of our previous reports [8][9][10][11][12].Change of the aromatic or heteroaromatic nuclei to acyl group e.g.acetyl group may be interesting and will introduce new information in this field.Considering the above facts, we have taken a project to synthesize some monosaccharide derivatives (e.g.D-glucose, D- mannose, L-rhamnose etc.) in the furanose and pyranose form and disaccharide (e.g.sucrose) containing acetyl (CH 3 CO) moieties in a single molecular framework and to evaluate their comparative antimicrobial activities using a variety of pathogens.

General experimental procedures
Melting points (mp.) were determined on an electrothermal melting point apparatus and are uncorrected.Evaporations were performed under diminished pressure on a Büchi rotary evaporator.IR spectra were recorded on a FT IR spectrophotometer (Shimadzu, IR Prestige-21) using KBr and CHCl 3 techniques.Thin layer chromatography was performed on Kieselgel GF 254 and visualization was accomplished by spraying the plates with 1% H 2 SO 4 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 CDCl 3 as a solvent.Chemical shifts were reported in δ 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 acetylation:
To a solution of the hydroxyl compound in anhydrous pyridine (1 mL) was added acetic anhydride at 0 ºC followed by addition of catalytic amount of 4-dimethylaminopyridine (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) acetic anhydride and 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.

Antimicrobial screening tests
Four Gram-positive bacteria viz.Bacillus cereus BTCC  Salmonella paratyphi AE 14613, Salmonella typhi AE 14612 and Shigella dysenteriae AE 14369 were selected for antibacterial potentiality test.For the detection of antibacterial activities, the disc diffusion method described by Bauer et al. [21] was followed.Nutrient agar (NA) was used as basal medium for test bacteria.Dimethylformamide (DMF) was used as a solvent to prepare desired solution (1%) of the compounds initially.The plates were incubated at 37 °C for 48 h.Proper control was maintained with DMF.Each experiment was carried out three times.For antifungal screening tests one human and three phytopathogenic fungi viz.Aspergillus niger, Alternaria alternata (Fr) Kedissler, Curvularia lunata (Wakker Boedijin) and Fusarium equiseti (Corda) Sacc were used.The antifungal activities were assessed by poisoned food technique [22] as modified by Miah et al. [23].Potato dextrose agar (PDA) was used as basal medium to test fungi.

Synthesis of pyranose acetates
For pyranose sugar acetates, our first effort was to prepare glucose pentaacetate (5).This was accomplished by reaction of powdered α-D-glucose with excess amount of acetic anhydride in presence of anhydrous zinc chloride at 90 ºC using literature procedure [16].In the subsequent step, we attempted to prepare tetra-O-acetyl-D-glucopyranose.Thus, acetylation of methyl α-D-glucopyranoside (6) afforded a white solid, mp.104-105 °C in 86% yield.The IR spectrum of this solid exhibited no band for hydroxyl stretching.It showed bands at 1730 and 1762 cm −1 corresponding to carbonyl frequency indicating the attachment of acetyloxy groups in the molecule.In the 1 H NMR spectrum, four threeproton singlets at δ 2.08, 2.05, 2.01 and 1.99 were assigned for the four acetyloxy methyl protons.This was also confirmed by its 13 C NMR spectrum where four carbonyl carbon peaks at δ 170.6, 170.1, 170.0 and 169.6 and four acetyl methyl carbon peaks at δ 20.7, 20.6 (×2) and 20.5 were observed.Hence the structure of the compound was assigned as methyl 2,3,4,6-tetra-O-acetyl-α-D-glucopyranoside (7).
To increase biological activities we were interested to synthesize acylated glucopyranosides with aromatic moiety.So, we prepared methyl 6-O-trityl-α-Dglucopyranoside (8) according to the literature procedure [18].Acetylation of triol 8 furnished a white solid (91%), mp.121-122 ºC.The IR spectrum of this solid showed a band at 1751 cm −1 corresponding to carbonyl frequency and exhibited no band for hydroxyl stretching.In the 1 H NMR spectrum, three three-proton singlets at δ 2.08, 1.99 and 1.73 were assigned for the three acetyloxy methyl protons.The H-2, H-3 and H-4 protons appeared considerably downfield [δ 4.94 (H-2), 5.44 (H-3) and 5.07 (H-4)] as compared to its precursor 8.These downfield shifts clearly indicated the attachment of acetyl groups at C-2, C-3 and C-4 positions.In the next step, acetylation of methyl α-Dmannopyranoside (10) gave a compound in 89% yield as a thick oil.Its IR spectrum showed a band at 1747 cm −1 for carbonyl frequency hence indicating the attachment of acetyloxy groups in the molecule.In the 1 H NMR spectrum, four three-proton singlets at δ 2.14, 2.09, 2.02 and 1.97 were assigned for the four acetyloxy methyl protons.The IR and 1 H NMR spectra of this compound were in complete agreement with the structure accorded as methyl 2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside (11).Finally, direct acetylation methyl α-L-rhamnopyranoside (12) afforded a white solid, mp.89-90 ºC.The IR spectrum of the compound showed a band at 1743 cm −1 corresponding to carbonyl frequency.In the 1 H NMR spectrum, a three-proton singlet at δ 3.36 and a three-proton doublet at δ 1.20 (J = 6.2 Hz) were due to C-1 methoxy and C-6 methyl groups, respectively.In addition, three three-proton singlets at δ 2.12, 2.02 and 1.95 were assigned for the three acetyloxy methyl protons.Based on the spectral analysis the structure of the compound was assigned as methyl 2,3,4-tri-O-acetyl-α-Lrhamnopyranoside (13).

Structure activity relationship (SAR)
In vitro antimicrobial study (section 3.4.1 and 3.4.2) revealed that these compounds (1-14) were more active against Gram-negative organisms than that of Gram-positive and fungal organisms.An important observation was that, acetylated sugars with five-membered furanose form are less effective against both Gram-negative, Gram-positive and fugal pathogens than that of the corresponding acetylated sugars with six-membered pyranose form.This is because of the slight distortion of furanose ring in the presence of 1,2-Oisopropylidene ring.But monosaccharides (5-13) in pyranose form with regular 4 C 1 or 1 C 4 conformation exhibited excellent antimicrobial potentiality.Again, acylated monosaccharides with pyranose form showed much better antibacterial potentiality than that of sucrose (disaccharide) octaacetate ( 14) due to the presence of five-membered furanose ring.

Conclusion
Thus, we have successfully synthesized acetylated furanose (2,4), pyranose (5,7,9,11,13) and sucrose acetate ( 14) derivatives.A comparative study of in vitro antimicrobial activities of monosaccharide (furanose and pyranose form) acetates with sucrose (disaccharide) acetates was carried out successfully.The structure activity relationship (SAR) study revealed that the acetate derivatives in six-membered pyranose form were more prone towards antimicrobial functionality than that of the corresponding acetate derivatives in five-membered furanose form and sucrose octaacetate (14).