S B N P Enzymatic Synthesis & Functional Characterization Fredrik Viklund Department of Biotechnology Stockholm Royal Institute of Technology Surfactants based on natural products — Enzymatic synthesis and functional characterization Copyright © by Fredrik Viklund ISBN --- is is the electronic version of the thesis. Compared to the printed version, it includes some minor typographical corrections. Royal Institute of Technology Printed in Stockholm AlbaNova University Center May Department of Biotechnology SE- Stockholm, Sweden Universitetsservice AB http://www.biotech.kth.se/ http://www.us-ab.se/ To Explorers Sammanfattning Tensider är molekyler som består av en vattenlöslig och en fettlöslig del. De spelar en viktig roll i produkter som rengöringsmedel, kosmetika, läkemedel och mat såväl som i många industriella processer. Tensider används i mycket stor skala vilket gör det viktigt att minska deras påverkan på miljön. Det kan åstadkommas genom att använda naturprodukter som råvaror, genom att förbättra tillverkningsmetoderna och genom att minska användningen av begränsade resurser som energi och lösningsmedel. Den här avhandlingen behandlar lipaskatalyserad syntes av naturprodukts- baserade tensider. Den omfattar också studier av de framställda tensiderna; dels som antioxidanter i oljor, dels som tensider för att öka lösligheten av läkemedel. Omättade fettsyraestrar av askorbinsyra framställdes genom katalys med Candida antarctica lipas B i t-amylalkohol och i joniska vätskor. Höga utbyten av askorbyloleat erhölls i en jonisk vätska som formgivits för att öka lösligheten av fettsyran, när reaktionen kördes under vakuum. Vi fann att askorbyloleat är amorft och att det är en bättre antioxidant än askorbylpalmitat i rapsolja. Polyetylenglykol (PEG)-stearat, PEG -hydroxystearat och en rad PEG -acyloxy-stearater framställdes i en vakuumdriven och lösningsmedels- fri uppställning med lipas B från C. antarctica som katalysator. Kritisk micellbildningskoncentration (CMC) och solubiliseringsförmåga uppmättes för PEG -acyloxy-stearaterna. Deras effekter på levande celler utvärderades i studier av hemolys och ”transepithelial electrical resistance”. Flera av PEG -acyloxy-stearaterna var utmärka solubiliserare för farmaceutisk användning och hade försumbara negativa effekter på levande celler även vid höga koncentrationer. Enzymatiska och kemisk-enzymatiska metoder erbjuder unika möjligheter att framställa tensider av hög renhet. Rena och väldefinierade tensider möjlig- gör nya tillämpningar och är viktiga för förståelsen av förhållandet mellan tensiders struktur och deras funktion. Abstract Surfactants are molecules that contain a water-soluble and a fat-soluble part. ey have important functions in products such as detergents, cosmetics, pharmaceuticals and foods as well as in many industrial processes. Surfactants are used on very large scale, which makes it important to decrease their impact on the environment. is can be done by starting with natural materials, by improving the synthetic methods and by reducing the use of limited resources such as energy and organic solvents. is thesis focuses on lipase-catalyzed synthesis of surfactants based on natural products. It also includes functional studies of the produced surfac- tants; as antioxidants in oils, or as surfactants to solubilize pharmaceuticals. Unsaturated fatty acid esters of ascorbic acid were synthesized with cataly- sis by Candida antarctica lipase B in t-amyl alcohol and in ionic liquids. High yields of ascorbyl oleate were obtained in an ionic liquid that was designed to improve the solubility of the fatty acid, when the reaction was performed under vacuum. Ascorbyl oleate was amorphous and was a better antioxidant than ascorbyl palmitate in rapeseed oil. Polyethylene glycol (PEG) stearate, PEG -hydroxystearate and a series of PEG -acyloxy-stearates were synthesized in a vacuum-driven, solvent-free system using C. antarctica lipase B as catalyst. Critical micelle concentration and solubilization capacity were determined for the PEG -acyloxy-stea- rates. eir effects on living cells were evaluated in studies of hemolysis and transepithelial electrical resistance. Several PEG -acyloxy-stearates were excellent solubilizers for pharmaceutical use and had negligible negative effects on living cells even at high concentrations. Enzymatic and chemo-enzymatic methods offer unique possibilities to synthesize surfactants of high purity. Pure and well-defined surfactants en- able new applications and are important for the understanding of surfactant structure-function relationships. Contents List of articles 8 Introduction 9 Fat-soluble antioxidants 9 Pharmaceutical solubilizers 10 Enzymatic synthesis 11 Solubility 13 Heterogeneous reaction systems 14 Mainly solid systems 14 Homogeneous reaction systems 16 Substrate modifications 17 Surfactants as a part of the solution 18 Surfactant-coated soluble enzymes 18 Other methods 19 Ionic liquids 21 Substrates of different polarities 23 Inhibition and oxidation 24 Solvents of the future 25 Water and drying 26 Water as a substrate 26 Water and enzyme activity 27 Overall water effect 28 Drying methods 29 Salt hydrates and salt solutions 29 Chemically acting desiccants 30 Molecular sieves 30 Vacuum 31 Distillation 31 Activated acyl donors and drying 32 Vinyl esters 32 Non-ideal systems 32 Surfactant purity 35 Selectivity 38 Chemoselectivity 38 Regio- and substrate selectivity 38 Stereoselectivity 42 PEG monoesters 42 PEG monoalkyl ethers 42 Borate esters 43 Ethoxylation of fatty acids 44 Environmental aspects 45 Natural products and biocatalysis 45 Natural surfactants 46 Biological surfactants 47 Natural raw materials 47 Genetically modified organisms 48 Natural and healthy 49 Other aspects 49 Functional studies 51 Ascorbyl esters 51 Antioxidative effect of unsaturated ascorbyl esters 53 Pharmaceutical solubilization 55 Surfactant effects on cells 56 PEG-esters of hydroxy fatty acids 57 PEG 12-acyloxy-stearates 59 Structure vs. function 60 Future studies 61 Acknowledgements 62 References 64 List of articles is thesis is based on the following publications that are referred to by their roman numerals. I. Viklund F., Alander J. and Hult K. () Antioxidative properties and enzymatic synthesis of ascorbyl fatty acid esters. Journal of the American Oil Chemists’ Society In press. II. Park S., Viklund F., Hult K., Kazlauskas R.J. () Ionic liquids create new opportunities for nonaqueous biocatalysis: Acylation of glucose and ascorbic acid. In Ionic Liquids in Green Chemistry, Prospects and Opportunities ACS Symposium Series In press III. Park S., Viklund F., Kazlauskas R. and Hult K. () Vacuum-driven lipase-catalyzed direct condensation of -ascorbic acid and fatty acids in ionic liquids. Manuscript IV. Viklund F. and Hult K. () Enzymatic synthesis of surfactants based on polyethylene glycol and stearic or -hydroxystearic acid. Manu- script V. Viklund F., Söderlind E., Hult K. and von Corswant C. () Properties of PEG -acyloxy-stearates and their use as pharmaceutical solubilizers. Manuscript 8 9 Introduction e work presented in this thesis and the accompanying articles has been performed within the Centre for Surfactants based on Natural Products (SNAP, ). e main goals have been design, synthesis and functional studies of new surfactants. e surfactants have been targeted towards two specific applications: fat-soluble antioxidants and pharmaceutical solubilizers. e syntheses were performed using lipase catalysis. Fat-soluble antioxidants e fats and oils we use in food are mainly isolated from plants. ey naturally oxidize and become rancid upon storage. e oils contain molecules called antioxidants that protect the oil from oxidation. e natural antioxidants in oils mainly belong to the group tocopherols, which are more commonly called vitamin E. e interest in using special or highly refined oils in food is growing. Prod- ucts often contain nutritionally important, but sensitive, polyunsaturated fatty acids. ese include ω- fatty acids such as linolenic acid, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and ω- fatty acids such as linoleic acid (see the chapter on functional studies for structures). Polyunsatu- rated fatty acids are often present only in low concentrations in their natural sources. In the concentration process, natural antioxidants may be removed from the oil. To protect the oil from oxidation, rancid taste and destruction of essential fatty acids, antioxidants must be added. Vitamin C is an inexpensive and efficient natural antioxidant. Unfortu- nately, it is only soluble in water and cannot be directly used in oils. A com- mon way to make vitamin C more soluble in oils is to react it with a fatty acid to form an ascorbyl fatty acid ester. e only commercially used ascorbyl fatty acid ester today is ascorbyl palmitate. Ascorbyl palmitate is frequently used as an antioxidant for oils but is limited by a relatively low solubility. One way to increase the solubility of the ascorbyl fatty acid ester is to use ascorbyl oleate instead of ascorbyl palmitate. Ascorbyl oleate, as well as ascorbic acid and oleic 8 9 Introduction Introduction acid used for its production, are chemically sensitive compounds. is makes ascorbyl oleate difficult to produce with traditional chemical methods. By using enzymatic catalysis we have successfully produced ascorbyl oleate. We found that enzymatic synthesis in organic solvents is feasible and studied some of its limitations (Paper I). We also studied the activity of ascorbyl ole- ate as