Chemical modification of sophorolipids towards a platform of new derivatives Elisabeth I.P. Delbeke1, K.M. Van Geem2, C.V. Stevens1 1 SynBioC Research Group, Ghent University 2 Laboratory for Chemical Technology, Ghent University

Physico-chemical properties Introduction Sophorolipids: Building blocks for chemical derivatization Renewable resource High production cost (2-5 €/kg) Non-pathogenic yeast Biosurfactant Starmerella bombicola Biological activity Physico-chemical properties High production quantity (400 g/L) Complex structure

Results 1. Synthesis of intermediate sophorolipid aldehyde Influence of fermentation conditions on methanolysis reaction Protection of the sugar head proved necessary Optimized ozonolysis conditions to prevent to formation of stable secondary ozonides

Results 2. Synthesis of innovative derivatives Intermediate sophorolipid amines Quaternary ammonium sophorolipids Sophorolipid amine oxides Bolaamphiphilic sophorolipids Most interesting derivatives: Quaternary ammonium sophorolipids with octadecyl chain

Results 2.1. Antimicrobial evaluation Performed by the Laboratory for Microbiology (Ghent University) Gram-positive strains: Gram-negative strains: Staphylococcus aureus Bacillus subtilis Escherichia coli Klebsiella pneumoniae Significant growth inhibition against Gram-positive strains: - 8 peracetylated quaternary ammonium sophorolipids - 3 deprotected quaternary ammonium sophorolipids MIC determination for active compounds against Gram-positive strains: Staphylococcus aureus Bacillus subtilis Enterococcus faecium Streptococcus pneumoniae Control: Gentamicin sulfate

Results (µg/mL) 5a 5b 6a 6b Gentamicin sulfate S. aureus 10 5 E. faecium B. subtilis S. pneumoniae 25 (µM) 5a 5b 6a 6b Gentamicin sulfate S. aureus 8 6 5 10 E. faecium 21 B. subtilis S. pneumoniae 52

Results Synthesis of deglycosylated derivatives Evaluation of influence carbohydrate head Synthesis of deglycosylated derivatives Hydroxylated and non-hydroxylated quaternary ammonium salts

Results Comparison of antimicrobial activity with quaternary ammonium sophorolipids Performed by the Laboratory of Pharmaceutical Microbiology (Prof. Tom Coenye, Ghent University) S. aureus ATCC 6538 MU50 (µg/mL) MIC MBC 5a 7,81 31,25 62,5 5b 250 6a 1,95 3,9 15,63 6b 9a 9b 11a 125 11b SL lactone SL acid >1000 S. aureus ATCC 6538 MU50 (µM) MIC MBC 5a 6,59 26,36 52,72 5b 6,36 204 50,91 6a 2,18 8,76 4,37 17,53 6b 2,09 4,18 16,73 9a 3,44 55,08 110 9b 3,21 51,28 103 11a 56,68 227 11b 26,33 105 211 SL lactone 45,40 90,79 SL acid >1607 Acetylated SL quat Deprotected SL quat Hydroxylated quat Non-hydrox. quat

Results 2.2. Transfection efficiency evaluation Performed by CEMCA UMR 6521 and INSERM UMR 1078 (Prof. Paul-Alain Jaffrès, Brest University) Formulation of liposomal solutions Only homogenous formulation for 6a and 6b Evaluation of influence carbohydrate head Formulation with DOPE nescessary Complexation of pDNA at different CR (charge ratio) 6a/DOPE 6b/DOPE 9a/DOPE 9b/DOPE 11b/DOPE 11b/DOPE LFM = lipofectamine

Results DNA transfection to different cell lines Evaluation of cell viability Transfection efficiency 6a/DOPE 6b/DOPE 9a/DOPE 9b/DOPE 11b/DOPE 11b/DOPE Cell vialbility 6a/DOPE 6b/DOPE 9a/DOPE 9b/DOPE 11b/DOPE 11b/DOPE LFM = lipofectamine

Conclusions Synthesis of a four different sophorolipid libraries accomplished: Sophorolipid amines Quaternary ammonium sophorolipids Sophorolipid amine oxides Bolaform sophorolipids Promising antimicrobial and transfection efficiency results Positive influence of carbohydrate head was demonstrated

Acknowledgements Prof. dr. ir. C. Stevens Prof. dr. ir. K. Van Geem Prof. dr. ir. G. Marin InBio Research Group Prof. T. Coenye Prof. P.-A. Jaffrès The research leading to these results has received funding from the Long Term Structural Methusalem Funding by the Flemish Government (grant number BOF09/01M00409)