Synthesis , Characterization and Physicochemical Analysis of Some Mannofuranoside Derivatives with Potent Antimicrobial Activity

We have synthesized protected mannofuranose as a glycosyl donor and some heterocyclic moieties as glycosyl acceptor. Coupling of glycosyl donor and acceptor by glycosylation, results in the formation of mannofuranoside derivatives. Antimicrobial potential of synthesized compounds were tested against five different human pathogenic bacteria and two fungi by using microdilution method. Interestingly, all synthesized mannofuranoside derivatives gave antimicrobial activity. Cumulatively, inhibitory concentration (IC 50 ) against bacteria were found to be in the range of 84.28-309.43 μg/ml, while, IC 50 against fungus were in the range of 0.59-3.82 mg/ml. Ampicillin and Fluconazole were used as positive control. Further, physicochemical parameters of synthesized mannofuranoside derivatives were analyzed using in-silico approach. Fortunately, none of our synthesized mannofuranoside derivatives gave violation of Lipinski’s Rule of Five. Thus, we can safely state that these synthesized mannofuranoside derivatives could be considered as potential anti-microbial drug candidates.


INTRODUCTION
Microbial food contaminations have been the cause of severe public health concern over the last few decades 1 hence antibacterial and antifungal agents are necessary for food preservation.Many researchers have focused their research on finding high potential antibacterial and antifungal agents 2 .
The potential of sugars as starting compounds for highly efficient syntheses of carbohydrate conjugates are now well recognized with regard to antibacterial, antiviral, antineoplastic, antiprotozoal and antifungal activity [3][4][5][6][7][8][9] .Heterocyclic chemistry has wide array of biologically active compounds isolated from natural sources or synthesized in the laboratory, such as coumarins [10][11][12] , quinolines 13,14 , triazoles 15 and phenolics 16,17 etc.They are also if present as a part of giant molecule, imparting significant activity to these molecules.From the previous studies, it is realized that heterocyclic and aromatic rings are coupled with carbohydrate to enhance the biological activity 18 like some acyl-substituted D-glucofuranose were discovered as more biologically active than D-glucofuranose. 19urthermore, when biologically active molecule is connected to another nuclei may possess excellentbiological activity. 20veral glycoconjugates such as glycolipids, glycoproteins or glycopeptides & glycosylated natural products are frequently being used as antimicrobial drugs and also emerging as anti-cancer drug candidates [21][22][23][24][25][26][27][28] .Encouraged by these results, we have synthesized some glycosides for antibacterial and antifungal evaluation.During the synthesis of glycosides, protection of a particular functional group of monosaccharides is not only necessary for the modification of the remaining functional groups but also useful during the synthesis of novel derivatives of great importance 29,30 .Glycosylation of alcohol-acceptor precursor as heterocycles, with O-(2,3,5,6-di-O-α-Dmannofuranosyl) trichloroacetimidate 36 , as donor precursor in the presence of catalyst trimethylsilyl trifluoromethanesulfonate (TMSOTf) to give glycosidic products 31 , further antimicrobial activity of synthesized compounds were evaluated using various strains of bacteria& fungi to determine Inhibitory Concentration (IC 50 ) [32][33][34][35] .

MATERIALS AND METHODS
All the synthesized compounds were routinely checked for their purity on silica gel 60 (E.Merck) TLC plates and their spots were visualized by charring the plates with 5% H 2 SO 4 in MeOH.Column chromatography on Merck silica gel (100-200 mesh) was used for the purification of synthesized compounds.Combined organic extract were dehydrated using anhydrous Na 2 SO 4 and dried solutions were evaporated under vacuum.Solvents used in column chromatography were purchased from E. Merck, sd fine chemicals and Qualigens.Reagents used in the synthesis were purchased from, Avara syntheses, Sigma Aldrich and Himedia.

Apparatus
Electronics India, model-934 used for the determination of Melting points.ES mass spectra were recorded in Merck M-8000 LCMS system or Micromass Quadro LCMS system and HR/EI mass were done on JEOL-600H at 70eV.Proton NMR and 13 C-NMR spectra were recorded on Bruker Avance DPX-300 MHz or Avance DPX-200 MHz FT Bruker spectrometers, using deuteriated solvents and Tetramethyl Silane as an internal standard.The chemical shift values from downfield to upfield in both proton NMR and 13 C-NMR spectra.For synthesized compounds, proton NMR data is recorded in the order; chemical shifts values in ppm (δ), multiplicities are indicated as bs (broadened singlet), s (singlet), d (doublet), dd (double doublet), t (triplet), q (quartet) and m (multiplet).Coupling constant 'J' reported in Hz.Perkin-Elmer's Spectrum RX I FT-IR spectrophotometer was used for recording IR spectra using KBr disc.Elemental analysis was carried out on Carlo-Erba-1108 instrument.

Antibacterial activity Determination of inhibitory concentration (IC 50 ):
Thedisk diffusion method 38 was used for the preliminary antibacterial evaluation of compounds C1, C2, C4-C8.The inhibitory concentrations (IC 50 ) of mannofuranoside derivatives showing inhibition in the preliminary tests, were determined by the microtitre plate technique using micro dilution method (Table-1) 39 Briefly, Staphylococcus aureus (ATCC 6538), Bacillus pumilis (MTCC 160), Bacillus subtilis (MTCC 441), Bacillus cerius (MTCC1305), Escherichia coli (ATCC 25923), obtained from NCIM NCL Pune, India were grown to mid-logarithmic phase and harvested by centrifugation, washed with 10 mM sodium phosphate buffer (SPB) at pH 7.4, and diluted to 2 x 10 5 colony forming units (CFU)/ ml in SPB containing 0.03% Nutrient broth (NB).Mannofuranoside derivatives were serially diluted in 250 µL of nutrient broth (NB) medium in 96-well microtitre plates to achieve the desired concentrations (25-400µg/ml) with bacterial inoculum (5 x 10 4 CFU per well).After incubation at 37 o C overnight, the IC 50 was taken of the synthesized compound that provides a 50% reduction in growth of bacterial strains compared to the growth in the control well.

Antifungal activity Determination of inhibitory concentration (IC 50 ):
The utilization of microplate bioassays, or broth microdilution tests, to measure the biological action of synthetic compounds against fungal pathogens has expanded in recent years.This method has been recognized as the most encouraging invitro bioassay for evaluating antifungal activity 40,41 .The antifungal strains A. flavus (MTCC 277) and T. viridens (MTCC 167) for the test were obtained from NCIM, NCL Pune, India.Mannofuranoside derivatives were serially diluted in 250 µl of SD medium in 96-well microtitre plates to achieve the desired concentrations (0.5-5 mg/ ml) with fungal inoculum (5 x 10 2 CFU per well).The invitro antifungal activities of the test compounds under investigation were also done by Poisons Food technique 42 and the technique was used with some modification by 43 using 0.5-5 mg of compounds per ml of Sabouraud Dextrose (SD) medium.The diameter of radial growth of the test fungi was measured after 2 to 3 days of incubation at 27±1 o C and expressed as percent mycelia growth inhibition following the formula given below:

Calculation of Physicochemical Properties
To check the physicochemical properties of synthesized mannofuranoside derivatives, Molinspiration property count instrument(http:// www.molinspiration.com/cgi-bin/properties)was utilized.Different parametersas topological polar surface area (TPSA), molecular weight, miLogP, the number of hydrogen bond donors, the number of hydrogen bond acceptors, number of rotatable bonds, and violations of Lipinski's rule of five 44 were estimated using Molinspiration tool [Table -2

Physicochemical properties prediction
Earlier, lead drug compounds fails at different clinical trials stages as a result of in appropriate pharmacokinetic profiling.But, now-adays to cut down these failures, physicochemical profiling of new drug candidates has become an important part of drug discovery.These analyses involve assessment of various molecular descriptors that would help in prediction of druglikeliness.There are various in-silico tools available to calculate the molecular properties of a compound to assess its pharmacokinetic profile.In fact, these in-silico tools have helped a lot to overcome the issues related to the failure of clinical trials 45,46 .
Molinspiration is a tool which was used in the present study to screen the pharmacokinetic property of mannofuranoside derivatives i.e., C1, C2, C4-C8.According to the Lipinski's Rule of Five (Lipinski et
Eight mannofuranoside derivatives were synthesized and IC 50 was determined for all derivatives.From the IC 50 value depicted in table-1 it can be concluded that mannofuranoside with quinoline as heterocyclic substituent (C6) is more potent than coumarin analogue (C4), Phenolic (C5), triazole (C7)and methyl furanyl analogue (C8).
In this study, synthesized mannofuranoside derivatives, were found to be as strong antifungal agents.In addition, these compounds inhibited few pathologically important gram positive and gram negative bacteria also.As they gave significant antifungal potential, these compounds could be further improved to form broad spectrum antifungal agents.In addition, none of these mannofuranoside derivatives gave violation of Lipinski's Rule of Five.Further studies would be needed to decipher the exact mechanism of anti-microbial/anti-fungal action of these derivatives.However, we can safely state that the present study has provided some potent antimicrobial drug candidates in the form of mannofuranoside derivatives.
Scheme 3. Catalysed glycosidation of coumarin derivatives with glycosyl donor

Table . 1: Antibacterial (IC 50 µg/ml) and Antifungal activity (*IC 50 mg/ml) of compounds C1, C2, C4-C8
al., 2001) studied mannofuranoside derivatives posses' drug like property [Table-2].Lipinski's Rule of Five is based on the observation that the majority of oral drugs have a molecular weight ≥5000, Log P value ≥5, hydrogen bond donor ≥5, hydrogen bond acceptor ≥10 and rotatable bonds ≥10.Any compound which violates more than one of these rules might have problem.Interestingly, none of our synthesized mannofuranoside derivatives gave violation of Lipinski's Rule of Five.Thus, we can safely state that these compounds could be considered as potential drug candidates.