Disassembly of Bacterial Biofilms by the Self-Assembled Glycolipids

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Disassembly of Bacterial Biofilms by the Self-Assembled Glycolipids Derived from Renewable Resources Yadavali Siva Prasad, Sandeep Miryala, Krishnamoorthy Lalitha, K Ranjitha, Shehnaz Barbhaiwala, Vellaisamy Sridharan, C. Uma Maheswari, Chakravarthy S Srinandan, and Subbiah Nagarajan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 02 Nov 2017 Downloaded from http://pubs.acs.org on November 3, 2017

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ACS Applied Materials & Interfaces

Disassembly of Bacterial Biofilms by the SelfAssembled Glycolipids Derived from Renewable Resources Yadavali Siva Prasad,† Sandeep Miryala,‡ Krishnamoorthy Lalitha,† K. Ranjitha,† Shehnaz Barbhaiwala,‡ Vellaisamy Sridharan,† C. Uma Maheswari,† C. S. Srinandan*‡ and Subbiah Nagarajan*† †

Organic Synthesis Group, Department of Chemistry and CeNTAB, School of Chemical and

Biotechnology, SASTRA University, Thanjavur-613401, Tamil Nadu, INDIA. ‡

Biofilm Biology Lab, Centre for Research in Infectious Diseases, School of Chemical and

Biotechnology, SASTRA University, Thanjavur-613401, Tamil Nadu, INDIA. Corresponding authors: C. S. Srinandan - Fax: +91 4362 264120; Tel: +91 4362 304270; E-mail: [email protected] Subbiah

Nagarajan

-

Fax:

+91

4362

264120;

Tel:

+91

4362

304270;

E-mail:

[email protected], [email protected] KEYWORDS: Renewable resource, Supramolecular assembly, Glycolipids, Surfactant, Biofilm, Pathogen.

ACS Paragon Plus Environment

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ABSTRACT More than 80% of chronic infections of bacteria are caused by biofilms. It is also a long-term survival strategy of the pathogens in a non-host environment. Several amphiphilic molecules have been used in the past to potentially disrupt biofilms, however, the involvement of multi-step synthesis, complicated purification and poor yield still remains a major problem. Herein, we report a facile synthesis of glycolipid based surfactant from renewable feedstocks in good yield. The nature of carbohydrate unit present in glycolipid influence the ring chain tautomerism, which resulted in the existence of either cyclic structure or both cyclic and acyclic structures. Interestingly, these glycolipids self-assemble into gel in highly hydrophobic solvents and vegetable oils, and displayed foam formation in water. The potential application of these selfassembled glycolipids to disrupt preformed biofilm was examined against various pathogens. It was observed that glycolipid 6a disrupts Staphylococcus aureus and Listeria monocytogenes biofilm, while the compound 6c was effective in disassembling uropathogenic E.coli and Salmonella enterica Typhimurium biofilms. Altogether, the supramolecular self-assembled materials, either as gel or as surfactant solution could be potentially used for surface cleansing in hospital environments or the food processing industries to effectively reduce pathogenic biofilms.


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INTRODUCTION Over the last few decades, there is a tremendous growth in development of materials from renewable resources, such interest is driven by awareness and increased concern about the environmental impact.1,2 Moreover, the limited availability of fossil feedstock and its adverse effect led to the introduction of biorefinery concept.3 Towards the aim of attaining sustainability, interest in renewable surfactants has been emerging over the past few decades.4,5 Nowadays, the use of synthetic surfactants is continuously growing at a rate of 3-4% every year.6 On this basis, a wide variety of surfactants including anionic, cationic and neutral surfactants have been developed and utilized for various applications.7,8 Recent examples of bio-derived neutral surfactants synthesized from renewable resources represent a commercial alternative to petrochemical based building blocks.9-11 Generally, natural surfactants comprise carbohydrates, amino acids, polyphenols and polycarboxylic acids as a hydrophilic part and fatty acids, terpenes and sterols as a hydrophobic part.10,11 They are used in a wide range of applications such as detergents, personal care products, pharmaceuticals and agrochemicals.12-14 In particular, carbohydrate based surfactants, also known as glycolipids are most important class of amphiphilic compounds that has distinct biological properties and electronic applications.15-17 For example, the Alkylpolyglucosides (APGs) that are extensively used to enhance the foam formation in detergents, self-emulsifiers, and agrochemical formulations.18-20 Regardless of their popularity, synthesis of APGs and their analogues encounter a synthetic challenge, that involves the use of complicated protocol, formation of mixture of products and purification steps.21,22 Within this context, the use of cellulose and hemicellulose for the production of APGs using various catalysts has been largely explored.23,24 Recently, cellulose has been directly transformed into alkyl glucoside surfactant in one pot by using ionic liquid media and sulfonic resin as


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catalyst.25,26 Beach and coworkers developed a new class of non-hydrolysable linear and cyclic C-glycoside surfactants starting from monosaccharide via nonulose precursor in a relatively green manner.27 Recently, a new synthetic method to obtain long chain alkyl gluronamide based surfactant composition from polysaccharide has been developed by Benvegnu and coworkers.28 In this method, further purification has to be performed to get pure alkyl sugar amide from long chain alkyl gluronamide based surfactant composition. By considering the synthetic challenges and surfactant behavior of various glycosides, the continuous development of carbohydrate based surfactants is of great interest.29 Moreover, glycolipid isolated from plant and marine source displayed anti-biofilm activity against various pathogens.30,31 Glycolipids are significant in biology and medicine,32-34 thus development of sugar-based non-ionic surfactant possessing amide functionality from renewable resource via a simple synthetic method is essential. Cardanol is a unique value-added chemical derived from a renewable raw material, cashew nut shell liquid (CNSL), comprised of a rich mixture of phenolic lipids: 5% of 3-(pentadecyl) phenol (3-PDP), 50% of 3-(8Z-pentadecenyl) phenol, 16% of 3-(8Z,11Z-pentadecadienyl) phenol and 29% of 3(8Z,11Z,14-pentadecatrienyl) phenol.35 Moreover, the structural features such as aromatic ring, hydroxyl group and easily accessible saturated and unsaturated hydrocarbon chains render it capable of developing advanced functional materials with exceptional properties.36 Microbial cell assemblies enmeshed in matrix polymers are called biofilms. Microbes predominantly survive as biofilms in their natural settings. Also, up to 80% of chronic infections are caused by the biofilm lifestyle of bacteria.37 Kolter and Greenberg have discussed the molecular switch, different signals, and the social interactions occurring in the biofilms that result in its ecological success.38 Importantly, biofilm bacteria are resilient to environmental stresses and antibacterial chemicals like sanitizers, antibiotics, chlorine, etc.39 In non-host


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conditions, pathogenic bacteria are known to persist on inanimate surfaces as biofilms, which act as reservoirs and potentially seed infections.40 Particularly, biofilms formed on the surface of pipelines in food industries could contaminate food and water.41 However, Bryers has documented the problems related to medical biofilms, persistence and their interactions with host, and the novel technologies to control medical biofilms.39 Nevertheless, disruption of a preformed biofilm before intervention of the antimicrobials is a superior strategy.36,42,43 Natural cues does exist to release cells in a biofilm, as the dispersal is a part of its development cycle. Different nutrients like citrate, glucose, succinate, glutamate, ammonium chloride, oxygen, nitric oxide, etc. are known to disperse biofilms.44,45 Besides, quorum sensing and the secondary messenger, cyclic-di-GMP signals the cells to disperse from a biofilm.46,47 Bio-based surfactants, particularly the rhamnolipid and lauroyl glucose are implicated in the dispersal of biofilm bacteria including the dispersal of interspecies biofilm.48,49 Palmitoyl glucoside, a glycolipid isolated from S. marcescens was also demonstrated as a potential anti-biofilm agent.50 However, in the present study, we report the synthesis and supramolecular assembly of glycolipids from renewable starting materials and their application as potential anti-biofilm agents. Our investigation furnishes an insight on the influence of supramolecular assembly of glycolipid on the disassembly of pathogenic biofilms. EXPERIMENTAL SECTION Materials and general methods. All reagents and solvents needed for the synthesis of glycolipids were purchased from Sigma Aldrich, Merck, Alfa Aesar, SRL, and Avra chemicals, and were used as such without further purification. LR grade solvents were used to purify the glycolipids and recrystallization, and distilled solvents were used, when necessary. Solvents used for gelation studies are of AR grade and double distilled water was used for surfactant studies.


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The reaction progress was monitored by thin-layer chromatography using pre-coated silica gel plates purchased from Merck and visualized by UV detection or p-anisaldehyde stain or using sulfuric acid spray or molecular iodine. Characterization methods. 1H- and 13C-NMR spectra for glycolipids and their precursors were recorded on a Bruker Avance 300 MHz instrument in either CDCl3 or CDCl3 with few drops of DMSO-d6 or DMSO-d6 or D2O at room temperature. Chemical shifts (δ) are reported in parts per million (ppm) with respect to internal standard TMS and coupling constants (J) are given in Hz. Proton multiplicity is assigned using the following abbreviations: singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m). Electrospray ionization mass spectra (ESI-MS) were carried out in positive mode with a Thermo Fisher LCQ Advantage Max. instrument by dissolving the solid sample in methanol. Synthesis General procedure for the synthesis of 2-(3-alkylphenoxy)acetohydrazides. 2-(3Alkylphenoxy)acetohydrazides, 3a-c were synthesized in two steps.10 First step involves the synthesis of esters 2a-c from phenol or 3-alkylphenol 1a-c and second step involves the conversion of esters to the corresponding hydrazides, 3a-c. Synthesis of methyl 2-(3-alkylphenoxy)acetates 2a-c. To a solution of phenol or 3-alkyl phenol, 1a-c (20.0 mmol) in acetone, methyl bromoacetate (20.0 mmol) and anhydrous potassium carbonate (40.0 mmol) were added, and the reaction mixture was refluxed for 8 h. After completion of the reaction, as identified using TLC, the reaction mixture was cooled to room temperature and 5% NaOH was added. The mixture was extracted with ethyl acetate and dried over anhydrous sodium sulfate, followed by the removal of solvent yielded the pure


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product. Compound 2a was isolated as pale yellow viscous liquid and compound 2b and 2c were isolated as white solids.10 Synthesis of 2-(3-alkylphenoxy)acetohydrazides 3a-c. To the solution of methyl 2-(3alkylphenoxy)acetate, 2a-c (1 mmol) in ethanol, hydrazine hydrate (2 mmol) was added and the mixture was heated under refluxed for 12 h. After completion of the reaction, as identified by TLC, the reaction mixture was cooled and the precipitated product was filtered and dried under vacuum. The crude product can be further purified by recrystallization in ethanol.10 Compound 3a. Isolated as white crystalline solid; mp: 114-118 ºC; yield = 91%. 1H NMR (300 MHz, CDCl3) δ = 7.76 (br, 1H), 7.32-7.37 (m, 2H), 7.04 (t, J = 7.2 Hz, 1H), 6.91 (d, J = 7.5 Hz, 2H), 4.59 (s, 2H), 3.94 (s, 2H);

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C NMR (75 MHz, CDCl3) δ = 167.2, 155.7, 128.4, 120.8,

113.1, 65.48. Compound 3b. Isolated as white amorphous solid; mp: 64-66 ºC; yield = 82%. 1H NMR (300 MHz, CDCl3) δ = 7.75 (s, 1H), 7.22 (t, J = 7.8 Hz, 1H), 6.86 (d, J = 7.8 Hz, 1H), 6.76-6.69 (m, 2H), 5.02-5.38 (m, 2H), 4.57 (s, 2H), 3.93 (s, 2H), 2.58 (t, J = 7.8 Hz, 2H,) 1.67-1.57 (m, 4H), 1.31-1.25 (m, 18H), 0.88 (t, J = 6.9 Hz, 3H),

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C NMR (75 MHz, CDCl3) δ = 168.8, 157,2,

145.1, 130.0, 129.5, 122.4, 114.8, 111.5, 66.9, 36.0, 32.6, 31.9, 31.8, 31.3, 29.7, 29.5, 29.4, 29.3, 29.2, 29.0, 27.2, 22.7, 14.12. Compound 3c. Isolated as white amorphous solid; mp: 90-92 ºC; yield = 93%. 1H NMR (300 MHz, CDCl3): δ = 7.75 (br, 1H), 7.21-7.26 (m, 1H), 6.86 (br, 1H), 6.75-6.68 (m, 2H), 4.57 (s, 2H), 3.93 (br, 2H), 2.61-2.56 (m, 2H), 1.69-1.55 (m, 2H), 1.44-1.26 (m, 24H), 0.88 (m, 3H); 13C NMR (75 MHz, CDCl3) δ = 168.9, 157.1, 145.2, 129.5, 122.4, 114.7, 111.4, 66.9, 35.9, 31.9, 31.4, 29.7, 29.3, 22.7, 14.13.


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2.4.2 General procedure for the synthesis of glycolipids 6a-f. To a stirred solution of glucose 4

or

galactose

5

(1.0

mmol)

and

acetic

acid

(0.1

mL)

in

ethanol,

2-(3-

alkylphenoxy)acetohydrazide 3a-c (1.2 mmol) was added, and stirring was continued under reflux condition until the completion of reaction (12-24 h). After completion of the reaction, as indicated by TLC, solvent was removed to yield the crude products 6a-f. Pure glycolipid as white solid was obtained by recrystallization using methanol. Compound 6a. Isolated as white amorphous solid; mp: 132-134 ºC; yield = 86%. 1H NMR (300 MHz, D2O): δ = 7.28 (t, J = 8.4 Hz, 2H, Ar-H), 6.98 (t, J = 7.5 Hz, 1H, Ar-H), 6.89 (d, J = 8.1 Hz, 2H, Ar-H), 5.07-4.53 (m, 2H, -O-CH2), 3.92 (d, J = 6.3 Hz, 1H, Sac-H Ano-H), 3.71-3.58 (m, 1H, Sac-H), 3.50 (dd, J = 12.3, 5.7 Hz, 1H, Sac-H), 3.34 (t, J = 9.3 Hz, 1H, Sac-H), 3.20 (dd, J = 12.9, 2.7 Hz, 1H, Sac-H), 3.13-3.03 (m, 2H, Sac-H).

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C NMR (75 MHz, D2O) δ = 170.6,

156.9, 129.9, 122.3, 114.6, 89.5, 76.8, 76.2, 70.4, 69.4, 65.9, 60.8. HRMS (ESI): m/z calculated for C14H20N2O7 [M+Na]+ = 351.1168; observed = m/z 351.4667. Compound 6b. Isolated as pale white amorphous solid; mp: 98-100 ºC; yield = 88%. 1H NMR (300 MHz, DMSO-d6): δ = 9.06 (s, br, 1H, -NH), 7.06 (t, J = 7.2 Hz, 1H, Ar-H), 6.73 (t, J = 9.3 Hz, 2H, Ar-H), 6.63 (s, 1H, Ar-H), 5.71 (s br, 1H, Sac-OH), 5.59 (s br, 1H, sac-OH), 5.34 (dd, J = 10.8, 5.4 Hz, 1H, Sac-H Ano-H), 5.31 (s, 1H, -NH), 4.76-4.49 (m, 1H, -O-CH2), 4.39-4.24 (m, 2H, -O-CH2, Sac-H), 4.05 (s, 1H, Sac-H), 3.81 (d, J = 3.3 Hz, 2H, Sac-H), 3.60-3.09 (m, 4H, Sac-H), 2.59-2.45 (m, 2H, -CH2*), 2.00-1.98 (m, 2H, -CH2), 1.49-1.24 (m, 22H, -CH2), 0.89-0.85 (m, 3H, -CH3).

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C NMR (75 MHz, DMSO-d6) δ = 171.5, 167.1, 157.7, 143.9, 129.6, 129.1,

121.1, 114.6, 111.6, 90.6, 78.0, 76.6, 71.0, 70.3, 65.8, 61.3, 54.8, 35.2, 31.9, 31.3, 31.1, 30.8,


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29.1, 29.0, 28.9, 27.4, 22.2, 13.8. HRMS (ESI): m/z calculated for C29H48N2O7 [M+Na]+ = 559.3359; observed = m/z 559.8671. * signals merged with solvent peak Compound 6c. Isolated as white amorphous solid; mp: 156-158 ºC; yield = 92%. 1H NMR (300 MHz, DMSO-d6): δ = 9.61 (d, J = 4.5 Hz, 1H, -NH), 8.81 (s, 1H, -NH), 7.16 (dd, J = 7.8 Hz, 1H, Ar-H), 6.94-6.65 (m, 3H, Ar-H), 5.75 (t, J = 3.8Hz, 1H, Sac-OH), 5.02-4.98 (m, 2H, -O-CH2, Sac-H), 4.95 (t, J = 5.4 Hz, 1H, Sac-H Ano-H), 4.59-4.34 (m, 2H, -O-CH2, Sac-H), 3.87-3.76 (m, 1H, Sac-H), 3.71-3.64 (m, 1H, Sac-H), 3.48-3.40 (m, 1H, Sac-H), 3.22-2.96 (m, 4H, Sac-H), 2.51 (m, 2H, -CH2*), 1.54 (s, 2H, -CH2), 1.24 (s, 24H, -CH2), 0.85 (t, J = 6.5 Hz, 3H, -CH3). 13C NMR (75 MHz, DMSO-d6) δ = 171.9, 167.6, 158.2, 144.4, 129.6, 121.6, 115.2, 112.1, 91.2, 78.6, 78.1, 77.1, 71.5, 70.8, 66.3, 61.8, 35.7, 31.8, 31.4, 31.3, 29.5, 29.3, 29.2, 22.5, 14.4. HRMS (ESI): m/z calculated for C29H50N2O7 [M+Na]+ = 561.3359; observed = m/z 562.0667. * signals merged with solvent peak Compound 6d. Isolated as white amorphous solid; mp: 142-144 ºC; yield = 90%. 1H NMR (300 MHz, DMSO-d6): δ = 11.29 (s, 1H, -NH), 7.30 (dd, J = 8.4 Hz, 2H, Ar-H), 6.99-6.86 (m, 3H, Ar-H), 4.97 (s, 2H, Sac-H, -O-CH2), 4.58-4.46 (m, 7H, -O-CH2, Sac-H), 3.72-3.45 (m, 3H, SacH). 13C NMR (75 MHz, DMSO-d6) δ = 170.5, 156.9, 129.9, 122.2, 114.6, 114,5, 90.3, 76.1, 73.2, 68.8, 68.3, 65.8, 61.2. Compound 6e. Isolated as pale white amorphous solid; mp: 134-138 ºC; yield = 83%. 1H NMR (300 MHz, DMSO-d6): δ = 11.30 (s, 1H, -NH), 9.66 (d, J = 4.2 Hz, 1H, -NH), 7.20-7.11 (m, 1H, Ar-H), 6.80-6.64 (m, 3H, Ar-H), 5.72 (t, J = 3.0 Hz, 1H, Sac-OH), 5.49 (d, J = 6.9 Hz, 1H, SacOH), 5.37-5.36 (m, 1H, Alk-H), 5.33 (t, J = 4.8 Hz, 1H, Sac-H), 4.99-4.69 (m, 2H, -O-CH2, SacH), 4.58-4.46 (m, 2H, -O-CH2, Sac-H), 4.39-4.28 (m, 1H, Sac-H), 4.22-4.15 (m, 1H, Sac-H),


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3.75-3.48 (m, 4H, Sac-H), 2.51 (s, 2H, -CH2*), 1.99-1.94 (m, 3H, -CH2), 1.54-1.21 (m, 18H, CH2), 0.85 (t, J = 6.6 Hz, 3H, -CH3).

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C NMR (75 MHz, DMSO-d6) δ = 172.0, 167.4, 158.3,

144.2, 130.5, 130.1, 129.4,121.2, 115.2, 112.092.0, 77.0, 73.9, 69.5, 69.4, 66.2, 63.5, 60.9,35.7, 32.4, 31.8, 31.6, 31.3, 29.6, 29.4, 29.2, 29.1, 28.7, 27.1, 26.8, 22.6, 14.5. *Signal merged with solvent peak Compound 6f. Isolated as white amorphous solid; mp: 142-144 ºC; yield = 90%. 1H NMR (300 MHz, DMSO-d6): δ = 11.25 (s, 1H, -NH), 7.18 (q, J = 8.1 Hz, 1H, Ar-H), 6.81-6.64 (m, 3H, ArH), 4.96 (t, J = 6.6 Hz, 1H, Sac-H, Ano-H), 4.94 (t, J = 7.5 Hz, 1H, -O-CH2), 4.55 (t, J = 7.8 Hz, 1H, -O-CH2), 4.48-4.45 (m, 2H, Sac-H), 4.32 (dd, J = 11.1, 5.1 Hz, 1H, Sac-H), 4.22-4.17 (m, 2H, Sac-H), 3.73-3.34 (m, 6H, Sac-H), 2.50 (s, 2H, -CH2*), 1.54 (s, 2H, -CH2), 1.25 (s, 24H, CH2), 0.85 (t, J = 6.0 Hz, 3H, -CH3).

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C NMR (75 MHz, DMSO-d6) δ = 169.2, 167.2, 158.3,

154.6, 150.7, 144.4, 129.6, 121.7, 121.2, 115.3, 112.1, 72.7, 70.7, 70.2, 69.5, 66.6, 64.9, 63.3, 35.6, 31.8, 31.4, 31.3, 29.5, 29.4, 29.2, 22.3, 14.4. * Signals merged with solvent peak Gelation Studies A known quantity of glycolipid was mixed with appropriate amount of solvent/oil in a sealed glass vial, and the entire content was heated until the solid was dissolved. By this procedure, the solvent boiling point becomes higher than that under standard atmospheric pressure. The resulting solution was allowed to cool slowly to room temperature, and gelation was visually observed by inverting the vial upside down. A gel sample that exhibited no gravitational flow in inverted tube was obtained and has been denoted as “G”. Instead of forming gel if it remains as solution at the end of the tests then it is referred as “S” (solution) and if it remains as precipitate,


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the system was denoted as “P” (precipitation). The system, in which the gelator is not soluble even at the boiling point of the solvent, it was called as an insoluble system (I).10 Morphological Study To obtain a clear overview of morphological feature of the organogel, optical microscopy and high resolution transmission electron microscopy (HRTEM) were carried out using Carl Zeiss AXIO ScopeA1 fluorescent/phase contrast microscope and JEOL JEM 2100 F HRTEM respectively. A small portion of organogel was placed on a glass slide and mounted on Phase Contrast Microscope to identify the morphology of the gel. For HRTEM analysis, gel formed in cyclohexane was further diluted with cyclohexane followed by drop casting on TEM grid. Rheological measurements The mechanical behavior of gel formed by glycolipid was identified using a stress controlled rheometer (Anton Paar 302 rheometer) equipped with a steel-coated 25 mm diameter parallelplate geometry. The gap between two plates for rheological testing of glycolipid gel was fixed as 1 mm and experiments were carried out at 23 °C. Amplitude sweep measurement furnishes the information about linear viscoelastic range which is directly proportional to the mechanical strength of the gel material. Measurement of the storage modulus, G’ and the loss modulus, G” as a function of frequency sweep from 0.1 to 300 rad s-1 furnishes the mechanical behavior of gel. Rheology of surfactant solution was also measured under above mentioned condition using Anton Paar 302 rheometer. Surface tension measurements


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Surface tension of glycolipid at the water-air interface was investigated using the pendant drop technique using Rame-Hart model 250 tensiometer with DROPimage advanced software. The entire experiment was carried out at 25 ºC using an environmental chamber. The image was acquired by built-in imaging system provided by Rame-Hart and further processed by image analysis software. Bacterial Strains and Culture Conditions Strains used in this work are uropathogenic Escherichia coli UTI89, Pseudomonas aeruginosa ATCC 27853, Salmonella enterica Typhimurium 14028, Staphylococcus aureus ATCC 25923, and Listeria monocytogenes MTCC 657. Lysogeny broth (LB) was used to maintain and grow the bacteria for most of the bacteriological experiments. However, biofilm experiments were performed with Yeast Extract-Casamino Acid (YESCA) medium for E. coli, Tryptic Soy broth (TSB) for S. aureus and L. monocytogenes, LB broth without NaCl for Salmonella, and LB medium for P. aeruginosa. The absorbance for measuring the planktonic and biofilm growth was monitored in Tecan SunriseTM Microplate Reader. The colony morphology of the strains for qualitatively determining matrix production was tested by adding 40 µg mL-1 of congo red dye and 20 µg mL-1 of coomassie brilliant blue with or without supplementation of 5% (w/v) of sucrose to the media that was used for biofilm experiments51-53. Biofilm Assay Quantitative biofilm development was measured by the method described by O’Toole and Kolter.48 Briefly, an overnight grown culture was diluted to 1:100 and inoculated in 200 µL of the medium containing different concentrations of the glycolipids in the microtiter well and was incubated for 24 h at 37 °C. The grown culture was aspirated and the wells were washed three


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times with 200 µL of sterile phosphate-buffered saline (PBS) to remove the non-adherent cells. Wells were then dried and the adherent biofilm was stained with 200 µL of 1% crystal violet (CV) for 15 min. The unbound CV was removed by rinsing thrice with 200 µL of PBS. Further, 70% ethanol was added and incubated for 15 min to de-stain the CV and the absorbance of each well was measured at 600 nm using the microplate reader as a proxy for the biomass. In order to study the disruption, the pre-grown biofilm was treated with the glycolipids 6a-c, sodium dodecyl sulfate (SDS), cetyl trimethyl ammonium bromide (CTAB) and chitosan (medium molecular weight) for 30 minutes, whereas PBS was added as a negative control. The wells were then aspirated and rinsed thrice with PBS before quantifying the biofilm biomass as mentioned above. Confocal Scanning Laser Microscopy (CSLM) The biofilm structure was analyzed by acquiring images of the biofilm formed on glass slides by CSLM (Olympus FLUOVIEW FV1000). Biofilm of 24 h old was developed on glass slides in the same way as described above. The media was decanted and slides were washed with PBS. The compounds were added on the slides and incubated for 30 min. Slides were then washed and stained using the BacLight Viability Kit (Thermo Fisher Scientific, L13152) according to the manufacturer’s instructions. Around 20 different fields were acquired randomly for a sample with same settings. The structural parameters from the biofilm images were quantified by COMSTAT program.54 Measuring the permeability of the cells The permeability of the cells was analyzed according to Niven and Mulholland.55 Briefly, 1mL of an overnight grown culture was harvested after centrifugation at 10000 g for 10 min and washed thrice with sterile PBS. The cell pellets were resuspended with an equal volume of PBS


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and 5 µL of propidium iodide (PI) as a negative control. For the test samples, the appropriate concentrations of the glycolipids, SDS, CTAB and chitosan were added along with 5 µL of PI. After 1 h of incubation, the fluorescence was measured using a Hybrid Multi-Mode Reader (Synergy H1 BioTek plate reader). Permeability index was calculated by normalizing the fluorescence value of the test samples to that of the negative control Results and discussion Synthesis The work presented herein represents an effort in generating a new class of glycolipid from readily available renewable resources, cardanol derivatives and monosaccharides by adopting simple synthetic protocols. The reaction of equimolar quantity of hydrazides, 3a-c with monosaccharides such as glucose 4 and galactose 5 in the presence of acetic acid and ethanol resulted in the formation of glycolipids, 6a-f in good yield under mild condition without the need of column chromatography purification (Scheme 1). This reaction is proposed to proceed via the well-established hydrazone intermediate followed by the subsequent cyclization step. Although previous effort to access a wide range of glycolipids have been made, their synthesis necessitates the use of complex and advanced starting materials, and intensive purification techniques.56-58 Glycolipids, 6a-f were characterized using NMR and mass spectral techniques.


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Scheme 1. Synthesis of glycolipids 6a-f. The existence of ring chain tautomerism i.e. cyclic form and acyclic form in glycolipids derived from monosaccharides were identified by the chemical shift of anomeric proton. In cyclic form, the anomeric proton resonates around δ 4.5-5.5 ppm, whereas acyclic isomer display signal for imine proton around δ 7.5-8.5 ppm with subsequent vanishing of signal for anomeric proton. 1H NMR spectral studies of glycolipids derived from glucose displayed exclusive formation of cyclic form, whereas in case of galactose ring chain tautomerism was observed, resulted in the existence of both cyclic and acyclic structures. This result suggested that the nature of monosaccharide determines the existence of cyclic or acyclic or both in solution form. The existence of β-anomeric form in cyclic structure was identified by calculating the coupling constant, J = 5.5-6.5 Hz for anomeric proton, which resonate at around 4.5-5.5 ppm. In the past few decades a detailed research on self-assembly has been perceived on inexpensive naturally occurring carbohydrate based polymers.59,60 In addition to the abundance and


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availability, the presence of hydroxyl groups, often used for further functionalization and hydrogen bonding probe the researchers to further investigate in the field of molecular selfassembly and supramolecular gels.59,60 The progress in this field resulted in the development of low molecular weight gels formed by self-assembly.61 Although low molecular weight gelators (LMWGs) were reported in nineteenth century, science behind the formation of gel by LMWGs was not clearly demonstrated.62 Shinkai and coworkers have generated monosaccharide based functional molecules and extensively studied their self-assembly mechanism and gelation behavior.63-65 Further, detailed study perceived by Shimizu and coworkers on glycolipid formed by monosaccharide with cardanol derivatives revealed the role of hydrophilicity, hydrophobicity and π-π staking in molecular self-assembly.66 Recently, Bhattacharya and coworkers discussed briefly on sugar derived LMWGs, which include their mechanism of gelation, structural analysis, stimuli responsive behavior, sensing properties, self-healing nature, and bio and nanomaterial synthesis.67 However, with the knowledge on self-assembly behavior of sugar based amphiphiles, we have designed and synthesized a class of glycolipids by incorporating the structural features such as hydroxyl groups, amide, aromatic and hydrophobic moieties that are known to promote molecular assembly. In a first study, low molecular weight gels were prepared by heating the glycolipids in an appropriate amount of solvent followed by cooling the resultant solution to room temperature. While cooling, molecules start to assemble in a suitable environment leading to the formation of gel. Among the various glycolipids tested, 6b and 6c acted as efficient gelators that self-assembled through non-covalent interactions resulting in the formation of fibrillary network structure in which solvent was trapped by surface tension. Gelation ability of glycolipid in various solvents and oils is summarized in Table S1. Glycolipid 6b and 6c displayed excellent gelation characteristics in hydrophobic solvents namely, cyclohexane, n-


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heptane, paraffin oil, vegetable oil and diesel with the critical gelation concentration of 0.6, 1.0, 0.8 and 0.6 % (wt/v) respectively. The thermal reversibility of the gel was confirmed by repeated heating and cooling cycle. Transition temperature of gel (Tg) derived from 6b and 6c in cyclohexane (0.6 % wt/v) was determined as 45 ºC and 49 ºC respectively, which gradually increases with increase in the concentration of gelator. Compounds 6a and 6d didn’t form gel in any of the solvent tested, and such behavior is attributed to the lack of maintaining hydrophiliclipophilic balance (HLB). However, increasing the lipophilicity of the glycolipid by introducing unsaturated and saturated alkyl chain substantially increases the gelation ability. While performing gelation test of compounds 6b and 6c derived from glucose in water, we have observed foam formation, which is stable for 6 h at room temperature. Galactose derived glycolipids 6e and 6f did not form foam in water and results obtained are also not consistent, such behavior is attributed to the existence of acyclic and cyclic form in solution. Morphological analysis The self-assembled supramolecular structure of glycolipids was examined by optical microscopy and HRTEM analysis. Optical microscopy image of organogel obtained from 6c in cyclohexane clearly explained the formation of entangled thin fibrous network (Figure 1a-f). HRTEM analysis of gel formed by 6c in cyclohexane further provided a detailed insight of gel morphology (Figure 1g-i). This result clearly depicted the formation of well-defined fibrous network of dimension ranging between 100-200 nm with void size of 500-800 nm. In order to understand the effect of solvent on molecular assembly, morphology of gel prepared by glycolipid 6c in n-heptane was investigated (Figure 1j and 1k). HRTEM revealed the formation of fibrillary network, in which the width of the fiber and their organization in gel is different from the gel obtained in cyclohexane. The different morphology formed by glycolipid 6c in


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different solvents shows the influence of solvents in molecular assembly. Since the solution of 6c in water forms a stable foam, we were curious to know the morphology by direct drop casting of dilute solution of 6c in water on the grid. A typical HRTEM image is shown in Figure 1l. The image shows that the solution consists of partially aligned micellar region with a network of nanofibers.


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Figure 1. (a-d) Optical microscopy image displaying the progression of gelation of compound 6c in cyclohexane with respect to time; (e,f) Optical microscopy image of xerogel formed from compound 6c in cyclohexane; HRTEM images of (g-i) gel obtained from compound 6c in cyclohexane, (j,k) gel obtained from compound 6c in n-heptane and (l) surfactant solution obtained from compound 6c in water. Rheological studies Rheological behavior of self-assembled supramolecular gels is important for their real-time applications, in particular thermo-reversibility and thixotropic property.35,68 Thus, viscoelastic nature of gel was independent of frequency sweep suggesting that it possesses good tolerance to external forces (Figure S17). Thermo-responsive rheological studies were performed to depict the stability of gel. Gel formed by 6c in cyclohexane retains both structural and mechanical integrity at elevated temperatures for more than three cycles (Figure S17). Step strain experiment depicts the exceptional mechanical behavior and structural reorganization of these gels. By applying 100 % strain on gel, both G′ and G′′ values were apparently decreased because of the disassembly process, whereas the fast recovery G′ and G′′ was observed by releasing the strain to 0.1 %. The repeated cycle of step strain experiment clearly arguing the reversible nature of gel and fast recovery of the mechanical property (Figure S17). In continuation to the rheological measurements of gel, we have measured dynamic rheology of surfactant solution of compound 6b and 6c.69 The oscillatory or frequency sweep measurements were carried out by fixing the shear rate corresponding to linear viscoelastic range (LVE).70 The variation of storage modulus (G′) and loss modulus (G′′) were monitored as a function of applied frequency at room temperature by using surfactant solution prepared from compound 6b and 6c in water at 5 mM concentration (Figure S18). Throughout the entire region of frequency sweep, loss modulus G′′


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is higher than the storage modulus G′ and both increases linearly with respect to the increase in frequency sweep. This trend is attributed to the increase in density of micellar aggregates, followed by organization. Surfactant studies While performing gelation studies, foam formation was observed with glycolipids 6b and 6c, which is stable for a long period of time. These glycolipids self-assemble themselves to form a gel in hydrophobic solvents and behave as a surfactant in water by diffusion into air-water interface and reduce the surface tension of the water. As concentration increases, surfactant migrates and fully occupy the air-water interface. After complete assembly of surfactant on airwater interface, the excess glycolipids present in the solution form micelle, after which there won’t be any prominent decrease in surface tension.71,72 Figure 2 represents the plot of surface tension against log of surfactant concentration. The careful analysis of Figure 2 represents the cmc value of glycolipids 6a-c. Compound 6b and 6c form micelle at 2.0 and 2.1 mM concentration respectively, whereas 6a displayed irregular fashion of surface tension behavior (Table 1). Among a series of compounds investigated, 6c reduces the surface tension of water to a larger extent, which can be attributed to the presence of saturated hydrophobic tail in its structure. The presence of unsaturation in the hydrophobic unit of compound 6b substantially reduces the surfactant behavior. Moreover, compound 6a behave as a non-surfactant molecule because of the absence of hydrophobic unit.


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Figure 2. A plot of surface tension vs log C for compounds 6a-c. Table 1 Calculated surfactant properties for sugar-based surfactants, 6a-c S.No 1 2 3

Compound 6a 6b 6c

CMC/mM 2.0 2.1

γmin/mNm-1 51 44

Anti-biofilm studies Bio-based surfactants and some natural products are shown to disrupt preformed biofilms of various pathogens.47,73 We initially tested the influence of glycolipids 6a, 6b and 6c on the formation of the biofilm for different pathogenic bacteria (Figure 3). The glycolipid 6a inhibited S. aureus biofilm at 200 µg mL-1 (P < 0.05) and 400 µg mL-1 (P < 0.001) concentrations, and E. coli biofilm at 400 µg mL-1 (P < 0.01) concentration (Figure 3a). Glycolipid 6b inhibited S. aureus biofilm at the concentrations of 200 µg mL-1 (P < 0.01) and 400 µg mL-1 (P < 0.01), while it reduced E.coli biofilm, but not significantly (Figure 3b). Interestingly, the glycolipid 6c inhibited biofilm of all the tested organisms at 400 µg mL-1 (P < 0.05) concentration (Figure 3c). Generally, biofilm bacteria are resilient to removal from the surfaces due to their robustness in the structure. We further examined the nonionic glycolipids 6a-c for disruption of preformed


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mature biofilm and compared with an anionic surfactant like SDS, a cationic surfactant like CTAB, and chitosan that permeabilize the membrane electrostatically. The sub-MIC concentrations of 400 µg mL-1 and 100 µg mL-1 for the glycolipids 6a-c and for SDS, CTAB, and chitosan were used respectively for the biofilm disruption experiments. Growth curves for all these concentrations are shown in Figure S19, where the glycolipids 6a-c insignificantly inhibit planktonic cell growth than SDS, CTAB and chitosan.

SA (a)

5.0

LM

UPEC

STm

*** *

4.5 4.0 3.5 3.0

**

2.5 2.0 1.5 1.0 0.5 0.0

(b)

3.0

** **

2.5 2.0 1.5 1.0 0.5 ND

0.0

(c)

2.5

*

*

*

2.0 1.5

** 1.0

*

*

0.5 0.0

0 (µg mL-1)

50 (µg mL-1)

100 (µg mL-1)

200 (µg mL-1)

400 (µg mL-1)


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Figure 3. Influence of the different concentrations of the glycolipid (a) 6a, (b) 6b, and (c) 6c on biofilm formation. The bars represent the biofilm biomass quantified by the crystal violet method at an absorbance of 595 nm. SA (Staphylococcus aureus), LM (Listeria monocytogenes), UPEC (uropathogenic E. coli), and STm (S. Typhimurium). Error bars represent 95% CI, n = 6. ND = Not Determined. Kruskal-Wallis test with Dunn’s multiple comparison was performed to analyze the data. Significance between 0 µg mL-1 with other concentrations are only depicted. (* P

Disassembly of Bacterial Biofilms by the Self-Assembled Glycolipids

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