Synthesis and Antimicrobial Activity of Amphiphilic Carbohydrate Derivatives

Diaminas N-monoalquiladas foram sintetizadas e tratadas com D-ribonolactona ou D-gliconolactona. As aldonamidas resultantes foram avaliadas para atividade antimicrobiana contra S. aureus, E. coli, M. tuberculosis and C. albicans. Duas hidrazidas foram também preparadas a partir da ribonoidrazida e suas atividades biológicas comparadas com aquelas de análogos amida. Todos os derivados da ribonolactona mostraram ter atividade antitubercular moderada, alguns foram ativos também contra S. aureus.


Introduction
Surfactants are amphiphilic molecules which are widely used in many industries. Although conventional nonionic surfactants can be produced in large scale from petrochemical raw materials, the increasing need for biodegradable and less toxic products has led to numerous studies of new sugar-based surfactants, which can be prepared from natural raw materials. [1][2][3][4][5][6][7][8][9][10][11] These compounds possess a carbohydrate hydrophilic head (mono-or oligosaccharide), and a hydrophobic tail, usually derived from a fatty acid. The two moieties can be directly linked via a functional group (ester, ether, hydrazine, amine, etc) or separated by a spacer (gemini surfactants). Carbohydrate surfactants are of great interest because they are not noxious for the environment. 12,13 Due to their functional properties they can be used in several areas, such as food industry (emulsion stabilization, foaming), 14,15 biology (extraction membrane proteins), 16 glycobiology, 17,18 immunology, 19 detergents, and cosmetology (non-alergic compounds).
Carbohydrate-derivated amphiphilic compounds have also been studied for their antimicrobial action. [20][21][22][23][24] These compounds can display two mechanisms of action. They can act as nonionic surfactants: the hydrophilic moiety of surface active compounds binds to the hydrophilic portion of the membrane via hydrogen bonds. The hydrophobic moiety is then able to penetrate the lipid bilayer structure, provocating a disorder in the permeability and fluidity of the membrane. 25 They can also act as inhibitors of enzymes involved in the biosynthesis of the bacterial cell wall. Galactofuranosyl 1 26 and galactopyranosyl derivatives 2 27 and 3 28 (Figure 1) bearing long alkyl chains displayed antitubercular activity.
In the last years the intensive use of antibiotic has lead to an increase of the emergence of resistant bacteria. 29,30 There is a growing need for new class of antibacterial compounds having different mechanism of action compared to existing drugs. The antibiotic action of several of these drugs involves binding to specific receptors or enzyme inhibition, against which bacteria developed strategies, like mutation or gene acquisition, leading to resistance. 31 The mechanism by which nonionic surfactants cause cell death involves an unspecific interaction with the bacterial membrane, difficulting the generation of resistance.
We report in this work the synthesis and biological evaluation of N-alkyl-amino aldonamides from D-glucono-1,5-lactone and D-ribono-1,4-lactone, as well as two hydrazones prepared from D-ribono-1,5-lactone. The influence of the hydrophilic portion of the molecule and the length of the spacer and of the alkyl chain on antimicrobial activity will be discussed.
Preparation of aldonamides 9a-d, 10a-d, 11a-d, 34 and 12a-c 35 was achieved by the nucleophilic addition of alkylamines 5a-d and 6a-d to D-glucono-1,5-lactone 7 or D-ribono-1,4-lactone 8 (1 equiv/mol) in ethanol for 20 h. After purification by crystallization or chromatographic column, all compounds were obtained in good to moderate yields (Table 1) and were characterized by IR and NMR spectroscopy. Spectroscopic data were in full agreement with those expected. 1 H NMR spectra of compounds 9a-d and 10a-d showed signals referring to the hydrophobic portion of the molecule between 0.8 and 3.0 ppm. Characteristic resonances corresponding to the carbohydrate moiety were observed between 4.0 and 5.3 ppm, while the diamine portion displayed signals in the 3-4 ppm region of the spectra. 13 C NMR spectra showed signals at ca. 174 (CO amide), 76-61 ppm (carbohydrate moiety), 14-50 ppm (alkyl chain and methylene carbons of the diamine). Hydrazones 14 and 15 were obtained by treatment of ribonohydrazide 13 36 with octanaldehyde and decanaldehyde in 93 and 80% yield respectively (Scheme 2). 1 H NMR spectra of both compounds showed signals corresponding to the alkyl chain (0.8-2.2 ppm) and to the carbohydrate protons (4.4-5.3 ppm). The fact that only one signal was observed at 7.7 ppm (CH=N) suggested that only the E isomer of the hydrazone was formed. 13 C NMR spectra showed signals at ca. 170 ppm (CO), 152 (CH=N), 74-67 ppm (carbohydrate mioety), 33-16 ppm (alkyl chain).  The minimum inhibitory concentration (MIC), concentration that inhibits the colony forming ability, was determined by incorporating decreasing concentrations of the tested compound to a culture of the bacteria and incubated at 37 ºC for 24 h (M. tuberculosis, E. coli) or 48 h (C. albicans). Rifampicin, penicillin-G (400000 UI mg -1 ), nystatin (5914 UI mg -1 ), and chloramphenicol were used as controls. The results are reported in Table 2.
Compounds 9b, 9c, 10a, and 10d, derived from D-ribono-1,4-lactone were active against the Gram-positive bacteria S. aureus. The most potent compounds were 9c and 10d (MIC = 25 µg mL -1 ). Compounds 9c, 9d and 10d were also the most active against M. tuberculosis (MIC = 25 µg mL -1 ). Compounds derived from D-glucono-1,5-lactone have previously been tested against E. coli, S. aureus and C. albicans 35 and were not tested against these organisms in this study. The referenced work showed that they were active against S. aureus (MIC = 10-50 ppm), compound 12c being the most active one. Due to their low solubility only a few derivatives could be tested against M. tuberculosis. The most active was 12b (MIC = 50 µg mL -1 ). Hydrazides 14 and 15 displayed low antitubercular activity (MIC = 100 µg mL -1 ) and were inactive against S. aureus in the tested concentrations. Compound 10a was the only compound with antifungal activity.
These results showed that Gram-positive bacteria S. aureus and M. tuberculosis were more sensitive to these amphiphilic compounds than Gram-negative E. coli. The sensitivity increased with the elongation of hydrocarbon chain, and the best results were obtained for compounds having an alkyl chain with more than 10 carbons. Grampositive bacteria are characterized by having as part of their cell wall structure peptidoglycan as well as polysaccharides. The hydrogen bonding between the cell wall and the hydrophilic moiety is therefore stronger than in Gram- negative bacteria. Having its polar head anchored in the membrane, the hydrophobic tail can interact with the lipid membrane, causing distortions leading to cell death. This mechanism would explain the similar antitubercular activity of ribonamide derivatives 9b and 10b and their analogues 11b and 12b (100 µg mL -1 and 50 µg mL -1 , respectively). In the same way, the length of the spacer does not interfere with the antibacterial activity, and similar results were obtained using 1,2-ethanediamine, 1,3-propanediamine or an hydrazide linker.

Conclusion
A series of amphiphilic carbohydrate derivatives were synthesized and evaluated as antimicrobial and antifungal agents. Almost all of them displayed a moderated activity against M. tuberculosis, and some of them were also active against S. aureus. Nonionic amphiphilic compounds having low antibacterial activity and emulsification potential (8 < HLB < 18) 36 are suitable compounds for cosmetic formulations, as they are less aggressive to skin and cause least disturbance in skin flora. 37

General methods
TLC were performed on glass plates coated with silica gel 60G (Merck). Detection was accomplished with iodine vapor, by spraying the plates with a solution of sulphuric acid in ethanol (20% v/v) or with a ninhydrin solution in ethanol (0.5% m/v), followed by heating at 120 °C. Column chromatography was carried out on silica gel (E. Merck 230-400 mesh). Solvents were purchased from Vetec Química and were distilled before use. Reagents were purchased from Aldrich and used without further purification. Melting points were determined on a Microquímica MQAPF apparatus and are uncorrected. IR spectra were recorded using a BOMEM-FTIR MB102 spectrometer. Optical rotations were measured with a Perkin Elmer 341 polarimeter, using a sodium lamp (λ = 589 nm) at 20 ºC. 1 H and 13 C NMR spectra were recorded on Bruker Advance DRX300 and DRX400 spectrometer. Elementary analyses were performed by the Central Analítica of Instituto de Química of Universidade de São Paulo, Brazil.

Synthesis
General procedure for the obtention of aldonamides A solution of the lactone (4 mmol) in ethanol (20 mL) or in a mixture ethanol/water 1:1 (10 mL) was added to a solution of the alkylamine (4 mmol) in ethanol (20 mL). The mixture was stirred at room temperature for 24 h and concentrated under reduced pressure. The residue was chromatographed on silica gel when oily or crystallized from water.        A microbiologic suspension from cultures previously incubated for 24 h (bacteria) and 48 h (fungi) was prepared in sterilized saline until a 25% transmittance reading was reached at λ = 580 nm (UV-Vis mini1240, Shimadzu ® , Japan). The standardized microbiological solution was prepared by serial dilution in sterilized saline, and the colonies counted in Tryptic Soy Agar (Acumedia ® , Canada), by plating technique. The dilution which presented 10 3 -10 4 CFU mL -1 was selected for further inoculation in Tryptone Soy Broth (Merck, Germany) (adapted from The United States Pharmacopoeia, 1985).

N-[3-(N-dodecyl)-3-aminopropyl]-D-gluconamide
Four mL of the seeded broth were added to 1 mL of the previously diluted solutions. Positive control consisted of 4 mL of the seeded broth, 10 µL of sterilized DMSO/Tween (1:2, v/v) and 990 µL of sterilized saline. To ensure the material remained sterile for the whole assay, a negative control was prepared with 4 mL of non-inoculated broth, 10 µL of sterilized DMSO/Tween (1:2, v/v) and 990 µL of sterilized saline. The MIC was evaluated by the turbidity of the broth after 24 h (bacteria) and 48 h (fungi) incubation. Tests were performed in triplicate.
The antitubercular activity against M. tuberculosis virulent strain H37Rv was determined in vitro. Stock solutions of tested compounds were prepared in DMSO. The mycobacteria was subcultured on Lowenstein-Jensen medium at 37 ºC for 3 weeks, followed by subculture in Middlebrook 7H9 broth medium at 37 ºC for at least 10 days, until bacterial density corresponding to a 1.0 McFarland turbidity standard was reached. The tests were performed through the microplate Alamar Blue assay. [39][40][41] The mycobacteria suspension were diluted 1:25 in Middlebrook 7H9 broth medium (4 × 10 5 mycobacteria/mL) and 100 µL of serial dilutions of compounds in the same medium. After incubation at 37 ºC for 6 days, 25 µL of a 1:1 (v/v) mixture of Alamar Blue reagent and 10% Tween 80 was added and the plates were re-incubated at 37 ºC for 24 h. A change in the colour from blue to pink was observed in the wells where the mycobacteria grew. The visual MICs were defined as the lowest drug concentration that prevented the colour change.