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 an antioxidant in rapeseed oil and found that it performed significantly better than the presently available ascorbyl palmitate (Paper I). As a way to improve the efficiency and lower the environmental impact of the synthesis, we also studied the reaction in a new type of solvents, ionic liquids. e best results were obtained in an ionic liquid that was designed to have an improved solubility of the fatty acids used (Paper II). We found that the reaction was very sensitive to oxidation but obtained good results when using ionic liquids that were synthesized and purified with special attention to impurities (Paper II and III). We also showed that the reaction could be efficiently performed in a larger scale and that the product could be isolated using only environmentally friendly solvents such as water, methanol and ethanol (Paper III) Pharmaceutical solubilizers e pharmaceutically active substances in drugs are often insoluble in water. New techniques are increasingly used to discover new drugs and the fraction of water-insoluble active substances can be expected to grow in the future. When a drug must be given to patients as an aqueous solution (e.g. when injected in the bloodstream), the water-insoluble active substance must be dissolved in water. is can be achieved by the addition of a surfactant. Many surfactants are efficient solubilizers for drugs but most surfactants have devastating effects on living cells. Destruction of red blood cells, hemo- lysis, is commonly caused already at very low concentrations. Only a small number of surfactants that are both efficient solubilizers and biologically ac- ceptable are presently used for pharmaceutical solubilization. However, they have been found to sometimes cause severe side effects. erefore, they are only used when their performance as solubilizers is so important that the risks they present can be tolerated. Taxol for example, is one of the most important anti-cancer drugs. Its active component paclitaxel is only very slightly soluble in water (Singla et al., ). In current commercial formulations for injection paclitaxel is solubilized with the surfactant Cremophor EL and ethanol. To reduce the risks of allergic shock or even death, anti-cancer treatment by injection of paclitaxel is usually combined with corticoid or anti-histamine treatment. Still, more than of the patients react with hypersensitivity, the main reason being Cremophor EL (Gelderblom et al., ).
10 11 Introduction Introduction
e reasons why solubilizers cause negative effects such as hemolysis or hypersensitivity are multifaceted. Due to current production methods, commercial solubilizers are complex mixtures. Some of the components in the mixture are efficient solubilizers, some induce negative side effects and some of the components influence the pharmaceutical effect of the active substance (Gelderblom et al., ). To improve pharmaceutical solubiliza- tion and to better understand its biological effects it is important to use pure surfactants. We designed several new surfactants targeted towards pharmaceutical solubilization. Pure surfactants were obtained by using enzymatic synthesis (Paper IV) or a combination of enzymatic catalysis and traditional chemical synthesis (Paper V). We found a new type of surfactant (polyethylene glycol mono--acyloxy-stearates) that showed very promising properties. Some of the surfactants presented were excellent solubilizers and displayed no nega- tive biological effects in studies on hemolysis and transepithelial electrical resistance (Paper V). Enzymatic synthesis e synthetic methods used in this work are based on enzyme catalysis. En- zymes are the molecules that facilitate chemical reactions in (and sometimes outside) all living cells. ey can also be isolated, purified and used as catalysts in chemical synthesis in all scales. e general subject of using enzymes in chemical synthesis is well covered in many books and review articles (Faber, ; Davis and Boyer, ; Koeller and Wong, ; Roberts, ). is thesis discusses aspects of enzymatic synthesis that are important for the synthesis of surfactants. e enzymatic syntheses in this work are catalyzed by a lipase from the yeast Candida antarctica (Anderson et al., ). e biological role of lipases is to catalyze the hydrolysis of ester bonds in fats. Lipases, however, accept a broad range of substrates and can be used to catalyze the hydrolysis or esteri- fication of a wide range of substrates (Gandhi et al., ; Gotor, ). ey are also relatively robust enzymes that function well in organic solvents and under other conditions that often must be used in chemical syntheses (Kastle and Loevenhart, ; Krishna and Karanth, ). A lipase-catalyzed esterification begins with the acylation step where the catalytic serine residue in the lipase reacts with a carboxylic acid. e acid forms an ester bond with the hydroxyl group of the serine and an acyl-enzyme intermediate is formed (Figure ., upper half ). In the following deacylation step, an alcohol attacks the acyl-enzyme complex, the product ester is released and the enzyme returns to its original state (Figure ., lower half ). Lipase- catalyzed hydrolysis proceeds analogously with the difference that the enzyme
10 11 Introduction is acylated by an ester, followed by deacylation using water. Note that the carboxylic acid (or ester) always binds to the enzyme before the alcohol (or water) enters the reaction. is determines much of the kinetics and selectivity of lipase-catalyzed reactions. O
R1 OH H2O acid water O acylation HO Enzyme R1 O Enzyme deacylation acyl-enzyme O HO R2 R1 O R2 ester alcohol Figure 1.1. Enzyme-catalyzed esterification.
Enzymes have naturally evolved to be highly efficient catalysts. Enzyme- catalyzed syntheses can be performed at low temperatures with high selec- tivity and little byproduct formation and are used in large-scale commercial production of for example enantiopure alcohols, acrylamide, aspartame and penicillins (Schmid et al., ). In this work, enzymatic synthesis was es- sential to produce high purity surfactants which were used in the following functional studies.
12 13 Introduction
Solubility
Any chemical reaction combining two reactants requires the reactant mol- ecules to be brought in close contact for the reaction to take place. Surfactants are usually synthesized from one hydrophilic and one hydrophobic group with very different physical properties. is makes mixing or dissolving the reactants difficult. Surfactant synthesis in this respect resembles the proverbial “mixing oil and water”. In a lipase-catalyzed esterification, a carboxylic acid reacts with the lipase and subsequently forms an ester when the acyl enzyme reacts with the al- cohol moiety (Figure .). us, for a reaction to occur, all three components – enzyme, acid and alcohol – must be able to reach each other. Enzymes have evolved in aqueous biological systems and are not soluble in organic solvents used in chemical synthesis. erefore, they are used as protein powders or more often immobilized on an inert carrier material. Immobilization im- proves enzyme stability, improves diffusion of substrates and products to and from the enzyme and facilitates the handling and separation of the catalyst. An immobilized or precipitated enzyme has a negligibly low diffusion rate. Of the three components interacting in esterifications catalyzed by an immobilized lipase, only the two substrates can have diffusion rates high enough to allow for significant mass transport. Comparing enzyme catalysis with homogenous catalysis that uses a small-molecule catalyst dissolved in the system, the insolubility of enzymes can be a handicap. Considering the ad- ditional difficulties of keeping both the hydrophilic and hydrophobic reactants dissolved, it comes as no surprise that solubility is one of the major practical problems facing enzyme-catalyzed surfactant synthesis. However, having a completely soluble reaction system is not an absolute requirement. e terms homogeneous and heterogeneous catalysis are often used for sys- tems where the catalyst and the reactants are in the same or different physical states. In the following discussion, homogeneous and heterogeneous systems refer to reaction systems where all or some of the reactants or products are
12 13 Solubility Solubility fully dissolved. e enzyme catalyst may be present as a solid (usually im- mobilized) or dissolved. Heterogeneous reaction systems Equilibrium reactions are controlled by the relation of the chemical activi- ties of the components. Chemical activity is considered as a measure of the availability of a component in the solution and it increases nonlinearly with the component’s concentration (Moore, ). e activity is zero when the component is not present and one when it is saturated in the solution. A poorly soluble substance that saturates the solution already at a low concen- tration thus has an activity of one. Despite its low concentration it affects the equilibrium strongly. In an equilibrium reaction, substrates are consumed and products are ac- cumulated until the reaction reaches equilibrium. If the saturation concentra- tion of a product is lower than its equilibrium product concentration, it will precipitate when it reaches the saturation concentration. e reaction will proceed with simultaneous precipitation of product until the depletion of reactants limits the conversion further. e product is thus effectively removed from the reaction equilibrium. is increases the reaction yield and can also simplify purification and workup procedures. If the solubility of a substrate is sufficiently low, it may limit the reaction rate because of its inability to saturate the enzyme. Some substrates, such as polysaccharides, have such low solubility in organic solvents that they practi- cally do not react at all. Mainly solid systems Enzymatic synthesis in heterogeneous liquid-solid systems has been ex- tensively reported, especially peptide synthesis. Peptide synthesis catalyzed by chymotrypsin gave good yields in organic solvents, such as hexane, even though the enzyme, both substrates and most of the product were undissolved (Kuhl et al., ). Peptides have also been synthesized in completely sol- vent-free eutectic mixtures, where the mixing of solid substrates themselves formed the liquid phase, resulting in yields of up to (Gill and Vulfson, ; Gill and Vulfson, ). e reactions mainly proceeded in the liquid fraction of the mixture and addition of a small amount of solvent improved the reaction rate and conversion (Lopez-Fandino et al., ). It may be noted that enzymes in some cases catalyze reactions in solid phase but with much decreased efficiency (Skujins and McLaren, ). e theoretical requirements for heterogeneous and solid systems have been analyzed by Halling and Jakubke with coworkers (Halling et al., ; Ulijn et al., ). e equilibrium yield is maximized by using a solvent in which
14 15 Solubility Solubility
the substrates and products have minimum solubility (Ulijn et al., ). For example, thermolysin-catalyzed synthesis of Z-Ala-Leu-NH in water (Z is benzyloxycarbonyl) reached yield (Halling et al., ). Lipase-catalyzed esterifications of sucrose, fructose and sorbitol with oleic acid all reached more than fatty acid conversion in aqueous phosphate buffer (Seino et al., ). ese results may seem surprising because of the intuitive, but incorrect, conception that hydrolytic enzymes will always act hydrolytically in the presence of water. Heterogeneous, non-aqueous, reaction systems have also been used for en- zymatic surfactant synthesis. Björkling et al. presented efficient solvent-free esterification of ethyl glucoside in melted fatty acid with monoester yields higher than (Björkling et al., ). e process was later scaled up (Adel- horst et al., ) and further developed to commercial production. Later work has exchanged the alkyl glucosides for more polar sugars and monosaccharide fatty acid esters have been produced in high yields using mainly solid phase reaction systems (Cao et al., ). However, little information is available on the feasibility of esterification of disaccharides and longer carbohydrates in heterogeneous systems. Bradoo et al. used a lipase from the thermophile Bacillus stearothermophilus to catalyze the esterification of ascorbic acid and palmitic acid in a solvent free system at - °C with yields reaching up to (Bradoo et al., ). e reaction was also successful in hexane using B. stearothermophilus lipase as well as when using lipase from Aspergillus terreus (Gulati et al., ). Comparing the production of ascorbyl esters in hexane, Gulati et al. reached and yield respectively for palmitic and stearic acid but only when using oleic acid. We show in Paper I that ascorbyl oleate is amorphous compared to the crystalline ascorbyl palmitate and it does not precipitate efficiently in hexane. e accumulation of product in solution could well be the cause for the low yields for ascorbyl oleate observed by Gulati et al. Product precipitation is not always beneficial. It has been observed that product precipitation of sugar esters in a mainly solid system caused the reaction rate to decrease, possibly by mass transfer problems caused by the precipitated product coating substrates or enzyme (Cao et al., ). In Papers II and III we noted that even though both substrates are soluble in the ionic liquid, the product, ascorbyl palmitate, precipitated on the enzyme beads as the reaction proceeded. is hindered mass transfer and slowed down the reaction. In a heterogeneous reaction system, efficient mass transfer between the phases is required as only the dissolved molecules are available for the reac- tion. As the reaction consumes the available substrate in solution, undissolved substrate must be transferred into solution or the substrate’s concentration
14 15 Solubility Solubility will drop as well as the reaction rate. us, for a reaction system using solid substrates to be competitive, the dissolution of the substrate must not be rate limiting. is was exemplified by Halling and coworkers in the enzymatic esterification of α-glucose with lauric acid in t-amyl alcohol (Flores et al., ). e concentration of dissolved glucose initially dropped as its dissolu- tion rate was lower than the reaction rate. By replacing the α-glucose with crystalline β-glucose or amorphous glucose, both of which have a higher rate of dissolution than α-glucose, the overall reaction rate could be increased. Homogeneous reaction systems Not all reactions work well in heterogeneous systems and throughout the years a number of schemes have been presented to increase the solubility of the substrates to enable more efficient catalysis. e most common approach is to use a solvent that dissolves the substrates while being inert and retaining enzyme activity. For the syntheses of many surfactants of commercial interest, such as sugar esters, finding this solvent is very difficult. Commonly used solvents for surfactant synthesis includet -butyl alcohol, t-amyl alcohol, acetone, acetonitrile and dioxane (Cao et al., ; Woudenberg-van Oosterom et al., ; Cao et al., ; Córdova et al., ) and Paper I. However, the solubility of some important substrates, such as glycosides, is limited in these solvents and much work has been done to find alternative reaction systems. Some solvents efficiently dissolve both hydrophilic and hydrophobic groups, e.g. dimethyl sulfoxide, dimethyl formamide and pyridine. Unfortu- nately they tend to inactivate enzymes by breaking hydrogen bonds that are essential for the structure and catalytic activity of the enzyme. Nevertheless, pure aprotic solvents such as pyridine (erisod and Klibanov, ) and di- methyl formamide (Riva et al., ) have been successfully used for enzymatic esterifications. Using solvent mixtures such as dimethyl sulfoxide andt -amyl alcohol have proven to increase the solubility of the substrates while being more tolerable for the enzyme (Ferrer et al., ). Many of the used aprotic solvents, e.g dimethyl sulfoxide, have an unpleasant odor and pungent taste and can be very difficult to completely remove from the product. is limits their use in food, cosmetical and pharmaceutical applications. In surfactant synthesis, dissolving the hydrophilic part is often the main solubility concern as the hydrophobic part is usually a fatty acid with good solubility in organic solvents. In Paper IV we esterified polyethylene glycol (PEG) with -hydroxystearic acid. e hydroxyl group gives -hydroxy- stearic acid limited solubility in most organic solvents as compared to stearic acid. PEG, on the other hand, has very favorable solubility properties and is
16 17 Solubility Solubility
freely miscible with melted -hydroxystearic acid. is enabled us to perform the esterification in a homogenous, solvent-free system. e melting point of -hydroxystearic acid is rather high ( °C) due to the free hydroxyl group. Exchanging -hydroxystearic acid for -acyloxy-stearates with lower melt- ing points in Paper V resulted in a system more tolerable for the enzyme. In a lipase-catalyzed esterification of polyethylene glycol monomethyl ether (MePEG) and -stearoyloxy-stearate, however, the reactants did not mix freely and the reaction was slower than for the other PEG esterifications where the reactants were completely miscible (unpublished data). Substrate modifications Derivatization of substrates is a technique often used in organic chemistry. Usually, this is used to improve the selectivity in a reaction as the derivatiza- tion block groups that are otherwise reactive. e strategy is therefore sometimes referred to as protecting group chemistry and is often used with carbohydrates. Sugars can be derivatized with acetals and then become more soluble in organic solvents. is has been used in enzyme-catalyzed esterifi- cation of monosaccharides where derivatization is used to improve substrate solubility and not to change the selectivity of the reaction (Fregapane et al., ). e method was later extended to allow for synthesis of disaccharide esters reaching yields of up to (Figure .) (Sarney et al., ). Not all sugars are equally well suited for acetal derivatization as the acetal formation depends on the positions and configuration of the hydroxyl groups (Evans et al., ; Greene and Wuts, ) and its application has so far been limited to mono- and disaccharides.
OH OH O O O O HO HO O 2,2-dimethoxypropane O O O O O O O HO OH O OH HO OH p-TSA O OH lactose lactose tetra-acetal
Figure 2.1. Derivatization of lactose with acetals. p-TSA is para-toluenesulfonic acid.
16 17 Solubility Solubility
e solubility of saccharides can also be improved by derivatization with organoboronic (e.g. phenylboronic) acids (Figure .). is strategy was effi- ciently used to react mono- and disaccharides with various acyl donors (Ikeda and Klibanov, ). OH OH O O O HO HO B O 2 O 4 H O HO B O 2 OH HO B OH glucose phenylboronic acid
Figure 2.2. Derivatization of glucose with phenylboronic acid.
Protecting group chemistry complicates the overall process by adding protec- tion, deprotection and potentially purification steps. is makes syntheses involving protective groups more laborious and expensive than is usually tol- erable for the relatively cheap surfactants. In more specialized applications, such as surfactants for cosmetic or pharmaceutical use, it can nevertheless be a viable route. Surfactants as a part of the solution Another route to solubilize the substrates was chosen by Skagerlind et al., who designed a microemulsion system for the synthesis of ethyl-D-glucoside decanoate (Skagerlind et al., ). At the outset, the fatty acid and its sodium salt acted as the surfactant and as the reaction progressed it was replaced by the product, keeping the mixture a microemulsion at all times. As all com- ponents in the system were soluble and microemulsions are characterized by high diffusion rates this allowed for fast reaction rates. is approach has been successful in many instances (Holmberg, ). Forming a stable microemul- sion requires a fine balance between the reactants and this may limit its gen- eral application in surfactant synthesis. In collaboration with the Institute for Surface Chemistry, we investigated the possibilities to esterify disaccharides in microemulsion. With the addition of a support surfactant, AOT (sodium ,-bis-(-ethyl-hexyl)--sulfo-succinate), a stable microemulsions capable of dissolving of disaccharide was designed. Due to the high water activity, no product was formed. To increase product formation, the microemulsion was dried to a water activity of . during the reaction. Still, only minute amounts of product were detected (Bell, ). Surfactant-coated soluble enzymes Instead of dissolving the substrates, the enzyme can be dissolved in organic solvents. Lipases, as most other proteins, are not soluble in organic solvents
18 19 Solubility Solubility
even though they have developed to act on lipids in hydrophobic environ- ments (Singer, ). Several methods based on covalent modification have been devised to improve their solubility (DeSantis and Jones, ). Active enzymes can be solubilized in organic solvents by using reversed micelles of a surfactant (Luisi, ). When only - molecules of the surfactant didodecyl glucosylglutamate were used per enzyme molecule, lipase from Pseudomonas fragi still catalyzed the enantioselective esterification of alcohols in dry isooctane (Okahata et al., ). At such low surfactant con- centration, reversed micelles were not formed but the enzyme was solubilized as the head groups of the surfactant interact with hydrophilic residues on the enzyme surface (Okahata and Ijiro, ). e method has been demonstrated with nonionic, cationic as well as zwitterionic surfactants (Okahata and Ijiro, ) and has been used with lipases, glycosidases and catalytic antibodies (Okahata and Mori, ). Chymotrypsin was solubilized in isooctane by the surfactant AOT (sodium ,-bis-(-ethyl-hexyl)--sulfo-succinate) and catalyzed the esterification of an insoluble amylose polymer with vinyl dec- anoate (Paradkar and Dordick, a; b; Bruno et al., ). e approach has also been used for esterification of mono- and disaccharides in several organic solvents (Tsuzuki et al., ). Dissolving the enzyme improves the mass transfer and increases the overall reaction rate. High conversion in reasonable time becomes possible in systems where no reaction can be detected without enzyme solubilization (Bruno et al., ; Tsuzuki et al., ). However, the product yield is still limited by solubility and equilibrium relations (Halling et al., ). If the product is more soluble than the reactants, the low reactant concentration may not be able to push the reaction very far to the product side. Separating the solubilized enzymes from the product mixture can also be a challenge. Other methods Solubility problems also affect chemically catalyzed syntheses. Some surfac- tant syntheses circumvent solubility problems by choosing alternative reac- tion routes. e commercial surfactants alkyl polyglycosides (Figure .) have hydrophilic groups consisting of polymerized carbohydrates. Alkyl polyglyco- sides are synthesized by heating glucose and the fatty alcohol in the presence of an acid catalyst (Weuthen et al., ). Starting with glucose instead of a polyglycoside avoids solubility problems as glucose is less crystalline and has better solubility in the fatty alcohol. A similar strategy is used for surfactants based on sorbitan esters where sorbitol and fatty acids are heated to yield the product (Stockburger, ). A common feature of most polymerization processes is that they produce a wide variety of components. e demand for
18 19 Solubility increased product purity is a main argument for enzyme-catalyzed surfactant syntheses.
HO O p-TSA n OH OH HO 90-120 ºC, vacuum HO OH glucose fatty alcohol
HO O n H O OH O 2
OH OH n
alkyl polyglycoside Figure 2.3. Synthesis of alkyl polyglycosides via polymerization.
20 21 Solubility
Ionic liquids
e industrial use of organic solvents is expensive in terms of chemicals, handling, disposal and environmental costs. erefore, many processes are being redesigned to replace the organic solvents by water, super-critical car- bon dioxide and other “neoteric” alternatives because of environmental and economical reasons (DeSimone, ). One of the more recent alternatives to organic solvents is ionic liquids. Salts that are liquid at room temperature have been known for a long time (Sugden and Wilkins, ; Hurley, ; Hurley and Wier, ) and have had their primary application in electrochemistry (Swain et al., ; Chum et al., ; Wilkes et al., ; Scheffler et al., ). Molten salts had been sparingly used as solvents in organic chemistry at somewhat higher temperatures (Ken- nedy and Buse, ) but it was when the dialkylimidazolium salts appeared (Wilkes et al., ) that organic synthesis at room temperature became fea- - - sible (Boon et al., ). e use of water-stable anions (e.g. BF and PF ) greatly increased their synthetic value (Wilkes and Zaworotko, ). Since then, ionic liquids are increasingly used not only to mean molten salts, but molten salts used as solvents at room temperature. eir use has increased dramatically in the last decade (Figure .). A review with some personal perspectives on their history is available (Wilkes, ) and recent progress in the green aspects of new solvents has been summarized with a view towards the future (Matlack, ).
20 21 Ionic liquids Ionic liquids
350 Ionic liquids Molten salts 300
250
200
150
100 Number of articles 50
0 1980 1985 1990 1995 2000 Number of articles
Figure 3.1. Annual number of articles and reviews in the CAplus database with the concepts “ionic liquids” or “molten salts” in the title or abstract.
Ionic liquids have some interesting properties as solvents. As they are salts, they have no vapor pressure and do not evaporate, they can be extracted with water and/or other solvents and their polarity and other properties can be engineered by modification of the participating ions. Research on synthesis in ionic liquids has been very extensive over the last few years and the subject has been thoroughly reviewed recently (Holbrey and Seddon, ; Welton, ; Wasserscheid and Keim, ; Gordon, ; Zhao et al., ). Some alkylimidazolium ions employed in ionic liquids are shown in Figure .. Ionic liquids can also be used in enzymatic synthesis (Kragl et al., ). eir most striking property is perhaps their ability to dissolve polar sub- strates such as amino acids (Erbeldinger et al., ) carbohydrates (Park and Kazlauskas, ) or ascorbic acid (Paper II and Paper III) while retaining the activity of the enzyme . Lipases not only retain their activity, but are often more stable and may be more catalytically active in ionic liquids than in organic solvents. For example, using EMIM·BF containing water, Candida antarctica lipase B displayed three times higher activity and had a half-life . times longer than in hexane. Furthermore, the half-life increased - times in EMIM and BMIM ionic liquids when the substrates vinyl butyrate and butanol were added (Lozano et al., ). Esterase from Bacillus stearothermophilus retained its activity and its half-life was increased to more than hours in BMIM·PF and BMIM·BF at a water activity of ., compared to a half-life of and hours in hexane and methyl tert-butyl ether respectively (Persson and Bornscheuer, ).
22 23 Ionic liquids Ionic liquids
Improvements in regioselectivity have also been seen for lipases in ionic liquids. Using lipase B from Candida antarctica in MOEMIM·BF, of the products were glucose monoacetate, compared to only in tetrahydro- furan, both at conversion (Park and Kazlauskas, ). O N N N N
1-ethyl-3-methylimidazol-1-ium 1-(2-methoxyethyl)-3-methylimidazol-1-ium (EMIM) (MOEMIM)
N N N N
1-butyl-3-methylimidazol-1-ium 1-sec-butyl-3-methylimidazol-1-ium (BMIM) (sBMIM)
N N
3-methyl-1-(2-pentyl)-imidazol-1-ium (2PentMIM) Figure 3.2. Examples of alkylimidazolium ions used in ionic liquids.
Substrates of different polarities e reaction between two substrates of very different polarity, as is required in surfactant synthesis, poses some special problems. Most organic solvents capable of dissolving carbohydrates and other polar compounds (e.g. tetrahy- drofuran, acetonitrile, acetone, dimethyl sulfoxide and pyridine) are suitable for dissolving nonpolar compounds as well. However, because they easily form hydrogen bonds, these solvents also disrupt the structure of most enzymes. is makes them unsuitable for enzymatic catalysis. On the other hand, many enzymes are stable in ionic liquids that often show good solubility of polar substrates. Ionic liquids designed to maximize solubility of carbohydrates do not dissolve nonpolar substrates such as fatty acids to a large extent, which was seen in our work on ascorbyl fatty acid esters. In Paper II and III we selected and designed ionic liquids not only to dissolve the ascorbic acid but also the fatty acid. In the ionic liquids with the highest solubility of ascorbic acid, the solubility of palmitic acid was low and the reactions resulted in relatively low conversions of -. Using the less polar ionic liquid sBMIM·BF the conversion was . With the aim to fur- ther improve the solubility of the fatty acid, we designed an ionic liquid with a larger nonpolar group, PentMIMIM·BF with which the reaction reached
22 23 Ionic liquids Ionic liquids
conversion. Despite the modified ionic liquid, ascorbyl palmitate was not fully soluble and deposited on the enzyme beads. e addition of hexane (- of total volume) or polypropylene beads ( of enzyme carrier) to the reaction system decreased the deposition of product on the enzyme beads and increased the yields (Table .). In a larger scale vacuum-driven esterification of ascorbyl oleate the isolated yield of ascorbyl oleate was . e product was recovered using dilution and precipitation with methanol/ethanol and water. is was done to maintain the green properties not only in the synthetic step but also in the product isolation (Paper III). As methyl and vinyl fatty acid esters are even less polar than free fatty acids they have very low solubilities in the ionic liquids capable of dissolving ascorbic acid. In preliminary tests, we found that the reaction rate of methyl esters was not significantly better than with free fatty acids. Because of their insolubility they were also impractical to use (unpublished data). Furthermore, the advantage of being able to remove formed water with high vacuum in ionic liquids reduces the need to use activated acyl donors. Table 3.1. Optimized acylation of ascorbic acid in ionic liquids. Data from Tables 1 and 2 in Paper III.
Solvent Hydrophobic Acylating Conversion a Yield b additive reagent (%) (%)
sBMIM·BF4 – Palmitic acid 43 40 sBMIM·BF4 20% hexane Palmitic acid 73 43 sBMIM·BF4 20% hexane Oleic acid 76 42 2PentMIM·BF4 – Palmitic acid 74 53 2PentMIM·BF4 20% hexane Palmitic acid 81 44 2PentMIM·BF4 20% hexane Oleic acid 83 65 c 2PentMIM·BF4 polypropylene beads Oleic acid n.d. 61 a Based on the disappearance of ascorbic acid b Based on the formation of ascorbyl fatty acid ester c 50% of the immobilized enzyme beads n.d. Not determined Inhibition and oxidation It has been shown that small amounts of silver ions present in ionic liquid prepared with common methods may inhibit lipases (Park and Kazlauskas, ). One way to avoid this is to filter the ionic liquids on silica gel and wash with sodium carbonate, which instead leaves traces of sodium ions (Park and Kazlauskas, ). Metal ions and bases are known to be efficient catalysts for the oxidation of ascorbic acid and ascorbyl esters in air ( Jung et al., ; Bisby et al., ). e yields in Table . are considerably lower than the
24 25 Ionic liquids Ionic liquids
conversions. is reflects that oxidation of either substrate or products occur in the reaction mixture. We minimized the oxidation with careful use of inert atmosphere but were not able to completely remove the difference between conversion and yield. Using t-amyl alcohol as solvent (Paper I), we did not detect any oxidation of the ascorbyl moiety and a probable reason for the fast oxidation in the ionic liquids is remaining metal ion impurities. is illustrates the importance of efficient purification of ionic liquids. Solvents of the future Ionic liquids present unique opportunities to tailor solvent properties to the application. ey are liquid over a large temperature range and their negli- gible vapor pressure enables the use of vacuum to easily remove volatile side products. Ionic liquids provide an interesting alternative for many difficult syntheses but it is a young technology and is not yet widely used in industry (Adam, ). Much more work has to be done before ionic liquids can be routinely used as solvents on a larger scale. Ionic liquids are still expensive and require efficient recycling to be justified. Methods for solvent purification and product isolation have to be further developed as ionic liquids cannot be distilled or evaporated. Ionic liquids are often presented as environmentally friendly solvents. ey can already be considered a “greener” alternative than traditional organic sol- vents, but production and utilization methods have to be further optimized before they are truly environmentally friendly (Nelson, ). It is beyond doubt that ionic liquids will be used industrially in special applications but it remains to be seen if they will become the green solvents of the future on a wider scale.
24 25 Water and drying
Water and drying
In an esterification reaction, a carboxylic acid and an alcohol condense to produce an ester and water (Figure .). In a transesterification reaction, the acyl part is introduced as an ester in the starting material and an alcohol is produced as the coproduct. O O
R2 R2 R1 OH HO R1 O H2O Figure 4.1. Esterification of a carboxylic acid with an alcohol.
Esterifications and transesterifications are analogous and water and alco- hols are equivalent when considered as substrates. Water, apart from alcohols, has a unique importance for enzyme structure and activity. Water as a substrate e thermodynamic equilibrium constant of a reaction, Keq is the ratio of the chemical activities of the products to those of the reactants when the reaction has reached equilibrium. In most cases, especially in dilute systems, chemical activity is proportional to concentration and a value of Keq can be calculated from the concentrations of reactants and products (Figure .).
a aproduct esteraproduct alcohol or water C Cproduct esterCproduct alcohol or water Keq = � Keq = aacyl reactantaalcohol reactant Cacyl reactantCalcohol reactant
Figure 4.2. Equilibrium equation for an esterification where a denotes chemical activity and C denotes concentration.
One consequence of equilibrium thermodynamics is that a reaction will al- ways proceed in the direction that changes the product and reactant concen- trations so that their ratio approaches Keq. When the desired product is the ester, as for the surfactants synthesized in the present work, the concentrations
26 27 Water and drying
of product and coproduct start at zero and will increase until the reaction reaches equilibrium. For esters of carboxylic acids, the equilibrium constant is in general within the range .- (Goldberg et al., ; Tewari, ; Tewari and Bunk, ). In aqueous systems, the concentration of water is close to . M and the re- actant concentrations are usually within the .- M range. Deprotonization of the acid may decrease its effective concentration further. Typical product concentrations will not reach more than . M under these conditions. At a reactant concentration of M , this corresponds to conversion and is not synthetically viable for most processes. In organic solvents, the water concentration can easily be decreased to - µM (- ppm) or lower. In reactions where water is a product, dry media can theoretically increase the product concentration six orders of magnitude as its absence pushes the equilibrium to product formation. With more moderate assumptions, drying a reaction system from a water activity of . to . increases the equilibrium product concentration times. is is one of the main reasons for removing the water (or alcohol) formed in ester synthesis. e relation between water content and water activity is not straightforward due to the non-ideal properties of water mixtures (Halling, ). However it can be reliably predicted in nonpolar as well as polar solvents using semi- empirical methods (Condoret et al., ; Voutsas et al., ). Water and enzyme activity e low water activity in organic solvents not only affects the equilibrium but also the catalytic activity of the enzyme (Sym, ; Skujins and McLaren, ). In a transesterification of triglyceride in hexane, . water was re- quired (Tanaka et al., ) and water-saturated hexane was used to avoid extraction of water from the immobilization gels that were used in similar reactions (Yokozeki et al., ). e loss of activity in dry systems is based on that water molecules are important for structural hydrogen bonds, stabiliza- tion of the transition state and to maintain the ionization of structural and catalytic amino acids (Klibanov, ). Most enzymes are active only at water activities distinctly higher than zero. For example, almond glucosidase is only active at water activities above . (Ljunger et al., ) and for alcohol dehydrogenase from ermoanaerobium brockii, the lower limit is . ( Jönsson et al., ). Enzymes that require high water activities may limit the possibility to favor product formation by the removal of water. To improve the productivity in such systems, the reaction can be started at a high water activity to benefit from the high reaction rate.
26 27 Water and drying Water and drying
e water activity can subsequently be lowered to increase the total yield (Ljunger et al., ; Svensson et al., ). One important reason for the success of lipases as biocatalysts is their re- tained catalytic activity at water activities approaching zero (Acker and Wiese, ). Enzymes may actually be stabilized and withstand higher temperatures in organic media with very low water activity than when water is present (Loncin and Jacobsberg, ; Zaks and Klibanov, ). Today, lipase-cata- lyzed reactions in dry organic solvents are common. For example, synthesis of dodecyl decanoate with Rhizomucor miehei lipase in hexane at water activity less than . has been reported (Valivety et al., a). Different enzymes have different optimal water activities and the catalytic activity measured in very dry media is usually lower than the maximum activ- ity. In, for example, the formation of octyl-β-glucoside catalyzed by almond glucosidase in octanol, the maximum activity was found in water-saturated solutions and it steadily decreased to reach zero at a water activity of . (Ljunger et al., ). In another study, the maximum activity of Lipozyme (immobilized Rhizomucor miehei lipase) was found around water activity . in several different solvents and decreased with - at water activities be- low . or above . (Valivety et al., b). e rate of synthesis of ascorbyl laurate in t-amyl alcohol catalyzed by Novozym (immobilized Candida antarctica lipase B), increased twofold almost linearly when the water activity was changed from one to zero (Luhong et al., ). e maximum activity of an enzyme may thus lie in any region of water activity – high, medium or low – and must be experimentally established. Overall water effect Water has two different roles in enzymatic catalysis. It affects the catalytic activity of the enzyme (structural water) and is a reactant or product (substrate water). At low water activities, enzyme catalysis may thus be slow either due to the low availability of reactant or to enzyme inactivation. In a recent study, the rate of transesterification catalyzed byRhizopus oryzae lipase in diisopropyl ether increased steadily with decreasing water activity. e rate of hydrolysis decreased at the same time, resulting in less hydrolysis at low water activity — the intuitively expected behavior. In contrast, the transesterification activity of lipase fromCandida rugosa decreased faster than its hydrolytic activity when the water activity decreased. Changing the water activity from . to . thus surprisingly increased the relative rate of hy- drolysis (Ma et al., ). us, the selectivity of a lipase, for transesterification or hydrolysis, can change in either direction when the water activity changes. Other enzyme selectivities also vary with water activity, for example the
28 29 Water and drying Water and drying
enantioselectivity displayed by some lipases (Kitaguchi et al., ; Jönsson et al., ). e effect of water activity on the catalytic activity and specificity of -en zymes is complex. It is however clear that water plays important roles as re- actant, for enzyme structure and function as well as for substrate and product solvation. e subject has been thoroughly investigated and several reviews are available (Halling, ; Anthonsen and Sjursnes, ; Halling, ). Drying methods Several means of removing water from enzymatic reactions has been pre- sented, each with its advantages and disadvantages. Salt hydrates and salt solutions are well suited for small-scale reactions where they provide a simple and effective means of controlling the water activity. Molecular sieves and other absorbing desiccants are convenient for creating very dry solutions in the laboratory but are often impractical on a larger scale. Vacuum and distil- lation techniques can be complex to use in the laboratory but are more easily applied in large scale and also work well for volatile coproducts other than water. Salt hydrates and salt solutions Many salts form hydrates and binds water in a defined way. A constant water activity can be maintained by coupling the water in a reaction system to the binding or release of water by a salt (Kuhl et al., ). e salt hydrate works as a water buffer, binding or releasing as needed to keep the water activity constant (Halling, ). e salt and the reaction systems can be connected either by direct addition of salt hydrate into the reaction solution or less ef- ficiently through the vapor phase. Saturated salt solutions also have a defined water activity (Greenspan, ) and can be used as water buffers (Goderis et al., ). Usually, water transfers through the vapor phase but the salt solution can also be mixed with the solvent. Other means of phase contact is also available, such as pumping the saturated salt solution through silicone tubing immersed in the reaction sys- tem, which improved mass transfer as compared to vapor phase equilibration (Kaur et al., ; Wehtje et al., ). Both the amount of water produced in a reaction and the capacity of the salt hydrate or salt solution can easily be calculated to determine the amount of salt required to maintain a constant water activity (Halling, ). When water is produced or consumed in the reaction, water activity must be continu- ously controlled throughout the reaction. If the components are equilibrated separately, mixing effects may change the total water activity (Halling, ). Despite these well-known facts, reports are often seen where the reactants
28 29 Water and drying Water and drying are separately equilibrated and no water activity control is applied during the reaction. Salt solutions and salt hydrates are incompatible with some commonly used reaction systems. In the esterification of ascorbic acid int -amyl alcohol using lithium chloride solution, the salt reacted with the solvent (Paper I). Many salts used to obtain low water activities react with alcohols which results in solvent transfer, reduced reaction volume and loss of water activity control. Chemically acting desiccants In traditional organic chemistry, chemically acting drying agents such as calcium sulfate (Drierite), aluminum oxide, alkali metals and phosphorous pentoxide are often used for drying (Burfield et al., ). Most chemically acting drying agents are not possible to use directly in enzymatic reactions because they react with the enzyme. Nevertheless, they can be used if kept separate from the enzyme, for example when drying refluxing solvent in a distillation system. Molecular sieves e most widespread drying agent for enzymatic reactions in laboratory scale is probably molecular sieves. Molecular sieves are zeolites, ceramic materials with microscopic pores of defined sizes. Contrary to common beliefs, molecular sieves are usually more efficient than chemically acting desiccants. Drying with molecular sieves reduced the water content to less than ppm for a variety of polar solvents such as dioxane, acetonitrile and t-butanol. In nonpolar solvents such as ethers and hexane, concentrations below . ppm were reachable, corresponding to ex- tremely low water activities (Burfield et al., ; Burfield et al., ; Burfield and Smithers, ; ; Burfield et al., ; Burfield and Smithers, ; ; Burfield, ; Burfield et al., ). Water is absorbed by molecular sieves with or Å pores. Sieves with a pore size of Å also readily absorb methanol. Because of their convenience and efficiency, many researches use molecular sieves to remove water or methanol in enzyme-catalyzed reactions. Molecular sieves also act as solid state acid-base buffers that can affect the ionization state of an enzyme. Molecular sieves caused up to -fold rate enhancements in transesterifications catalyzed by subtilisin Carlsberg in supercritical carbon dioxide and polar as well as nonpolar organic solvents. Excessive amounts of molecular sieves slowed the reaction and addition of a second solid state acid-base buffer neutralized the effect of the molecular sieves (Fontes et al., ).
30 31 Water and drying Water and drying
Vacuum In a gas-liquid system, the partial pressure of a component in the gas phase (its vapor pressure) is proportional to its activity in the solution (Moore, ). If the pressure of the component is reduced in the gas phase, it will evaporate from the liquid to the gas phase until the system is in equilibrium again. is principle is the basis for drying using reduced pressure. e technique is very effective in reducing the water activity of a system. e vapor pressure of water is approximately mbar at °C and this is the pressure theoretically attainable with a laboratory water aspirator at that temperature. In a reaction system kept at °C, the equilibrium water vapor pressure is mbar and if the °C aspirator is connected to the reaction, the water activity in the reaction system is consequently reduced to /≈.. Using a vacuum pump, that does not saturate the atmosphere with water, it is possible to reach pressures well below . mbar corresponding to water activities of . or less. Using vacuum to directly remove water is very simple and efficient but it is only useful if water is the only volatile component in the system. It is therefore preferably used in solvent-free reactions (Adelhorst et al., and Paper IV). e only class of solvents that can be effectively used in vacuum systems is ionic liquids (Paper III). Ionic liquids have a very low vapor pressure, do not evaporate and can be tailored to suit the application (Kragl et al., ; Papers II and III). Ionic liquids present new opportunities to use simple dry- ing techniques in large scale which may decrease the cost and environmental impact of volatile solvents. Distillation If direct vacuum drying can not be performed, water can be removed by means of fractional distillation. Vapor is then evaporated from the reaction medium and water is removed from the vapor mixture using standard distillation techniques (Grüning and Hills, ; Humeau et al., ), a semi-perme- able membrane (Yan et al., ) or by absorption to a desiccant (Sarney et al., ). To achieve efficient mass transport, drying by distillation is performed in boiling reaction systems. e boiling point of the mixture may be lowered to temperatures tolerated by the enzyme by using reduced pressure or azeo- tropic solvent mixtures (Sarney et al., ; Yan et al., ).
30 31 Water and drying Water and drying
Activated acyl donors and drying To increase reaction yields and rates, activated acyl donors are used in lipase- catalyzed esterifications (Faber and Riva, ). Vinyl esters are the most versatile and frequently used alternative. Vinyl esters Vinyl esters are accepted as substrates by lipases (Brockerhoff et al., ). e released product vinyl alcohol immediately tautomerizes to acetaldehyde. e tautomerization is energetically very favored and the conversion to acet- aldehyde is practically complete. ere is thus no possibility for the enzyme to catalyze the backwards reaction and the esterification is pushed to almost yield (Degueil-Castaing et al., ). Vinyl esters are most frequently used but the same effect can be obtained with other enol esters such as isopro- penyl acetate (Wang and Wong, ). e use of enol esters can in some cases cause enzyme deactivation and loss of selectivity (Berger and Faber, ) As an esterification reaction proceeds, the product ester starts to compete as an acyl donor. If the enzyme is acylated by the product, there is no net product formation. Instead, the reaction slows down and the selectivity of the system decreases. Vinyl esters have high specificity constants and compete efficiently as substrates. Accordingly, high rate and specificity are obtained by using vinyl esters (Öhrner et al., ; Martinelle and Hult, ). In most systems there is a small amount of water present that can be used by the enzyme to hydrolyze either the vinyl ester or the product ester. Lipase-catalyzed hydrolysis of vinyl acetate was up to three times faster than product formation even under dry conditions where no hydrolysis of product was observed (Weber et al., ). Hydrolysis of vinyl ester results in drying of the reaction system and release of free acid. e synthesis will not reach yield unless an excess of vinyl ester is used. Non-ideal systems e high equilibrium yields predicted with molecular sieves are not always easily obtained. e product yield reached almost when glucose was esterified with C -C fatty acids in three times excess, catalyzed by lipase B from Candida antarctica in acetone (Arcos et al., ). When equimolar amounts of glucose and lauric acid were esterified using the same enzyme in t-amyl alcohol, the maximum yield reached only (Flores et al., ). e lower yield was attributed to the presence of water despite the use of molecular sieves. In Paper I we used molecular sieves to carefully dry the esterification of ascorbic acid with an equimolar amount of palmitic acid in t-amyl alcohol. e reaction reached only yield. e unexpectedly low yields could be ex-
32 33 Water and drying Water and drying
plained by a contaminating alcohol in the solvent, acting as a second substrate and lowering the yield of the ascorbyl ester. To test this hypothesis, t-amyl alcohol, vinyl stearate, molecular sieves and enzyme were mixed. e solvent was subsequently distilled to eliminate any products and was then dried over molecular sieves for six days. e purified t-amyl alcohol, however, failed to display better yields than the one used as received (unpublished results). Table . summarizes reports on enzymatic esterification of ascorbic acid in t-butanol, t-amyl, hexane and acetone that used molecular sieves.
Table 4.1. Yields in enzymatic esterification of ascorbic acid. Bradoo et al. used immobilized lipase from Bacillus stearothermophilus. Yan et al. used Chirazyme L-2 and the other studies used Novozyme 435, both being immobilized lipase B from Candida antarctica.
Solvent/ Ratio a T Pore size Yield Source Acyl donor (°C) (Å) (%)
t-Butanol Phenylbutyric acid 1:1 60 3 15 Otto et al., 1998
Acids C12-C18 6:1 45 4 57-38 Stamatis et al., 1999 Vinyl esters C8-C16 2:1 40 3 63-90 Yan et al., 1999 Vinyl palmitate 1-3:1 40 3 77-91 Yan et al., 1999
t-Amyl alcohol Methyl lactate 1:1 70 4 10 Maugard et al., 2000 Methyl lactate 2:1 70 4 19 Maugard et al., 2000 Methyl lactate 4:1 70 4 40 Maugard et al., 2000 Methyl lactate 8:1 70 4 60 Maugard et al., 2000 Methyl lactate 16:1 70 4 82 Maugard et al., 2000 Methyl lactate 20:1 70 4 84 Maugard et al., 2000 Palmitic acid 1:1 60 3 71 Paper I Palmitic acid 2:1 60 3 86 Paper I
Hexane Palmitic acid 1:1 50 — b 92 Bradoo et al., 1999 Acetone Vinyl esters C8-C16 2:1 40 3 55-76 Yan et al., 1999 Vinyl palmitate 1-3:1 40 3 60-86 Yan et al., 1999 a Ratio of acyl donor to ascorbic acid. b Pore size of the molecular sieve not given.
e yields are widely scattered despite the apparent similarities between systems. e low yields for methyl lactate and phenylbutyric acid can partly be explained by their bulky structures. e results obtained with methyl lactate
32 33 Water and drying
(Maugard et al., ) however fits well to an equilibrium equation and indi- cate that the reaction reached equilibrium. Reactions with vinyl acyl donors are practically irreversible and should give the same yield () independent of the acyl chain length but varied between and (Yan et al., ). ese reactions were performed in a mainly solid system and the polarity of the vinyl esters may affect the system more than in a system where solvent dominates. e fact that the yield improved with larger excesses of acyl donor indicates that hydrolysis of vinyl ester was present in the reaction. e differences may also be partly explained by the inefficient drying. -Dry ing with molecular sieves was slow and inefficient fort -butanol and -butanol compared with methanol and ethanol. Using powdered molecular sieves, the available surface area is larger and less than ppm was reached in hours for t-butanol (Burfield and Smithers, ). e poor drying of the C -alcohols was attributed to their high viscosity, which would indicate similar problems for t-amyl alcohol. e reasons for the variation and somewhat low yields seen in Table . can not be definitively determined but it seems reasonable that inefficient drying of tertiary alcohols with molecular sieves is the main reason.
34 35 Water and drying
Surfactant purity
e purity of a product can be defined in many ways. e most commonly used measure of purity in scientific publications is the fraction of a defined substance in the isolated product. When determining the purity of a surfac- tant, surface-chemical properties of the contaminants must also be taken into account. For example in measurements of surface tension and adsorption, it can be very important to avoid contamination of surface active species while contaminants that are not surface active can be present in considerable con- centration without affecting the characteristics of the product much. Polyethylene glycol (PEG) is commercially produced by polymerization of ethylene oxide. e process results in a polydisperse mixture consisting of a distribution of molecules of varying chain length. erefore, surfactants based on PEG are usually also polydisperse. Specified PEG chain lengths usually refer to an average of the distribution. It is sometimes useful to study surfactants with a specific chain length e.g.( CE, octaethylene glycol monododecyl ether). ere are synthetic methods to prepare monodisperse PEG with up to at least twelve ethylene oxide units (Keegstra et al., ) but the cost and difficulties are much greater compared to synthesis of polydisperse PEG. In this study we have used commercial, polydisperse PEG with average chain lengths of . (PEG) and (PEG) units (Paper IV and V). e reasons are convenience, economy and to get better comparisons with commercial alternatives. However, the methods presented for surfactant synthesis and study would work equally well with monodisperse or narrow-range distribution PEG. Many of the commercial surfactants currently used in food, cosmetics and pharmaceutics contain considerable amounts of free hydrophilic polymers (e.g. PEG, sorbitan or glycosides) that are not surface active. e hydrophobic reactants used in surfactant synthesis, on the other hand, are often surface ac- tive and may affect the system properties as well as being difficult to separate from the product mixture. erefore, surfactant syntheses are usually designed
34 35 Surfactant purity Surfactant purity to avoid remaining hydrophobic reactants. However, there are examples where remaining hydrophobic reactant in the product mixture improves its proper- ties e.g. alkyl polyglycosides that contain as much as free fatty alcohol (Weuthen et al., ). For use in food and personal care products, it is imperative to avoid bad taste and important to have a pleasing color and transparency. Surfactants used in pharmaceutical applications must not have adverse side effects when administered. While byproducts may complicate formulation and certifica- tion, high purity in itself is not an absolute demand in most food and phar- maceutical applications. e biological origin of enzymes can also affect the application of enzy- matic synthesis, especially in pharmaceutical products. e catalyst may be contaminated by lipopolysaccharides from bacterial cell walls released in the fermentations used for enzyme production. Some of the lipopolysaccharides, called endotoxins, can be extremely toxic and must be avoided. is problem is not limited to enzymatic catalysis of surfactants and is much more pro- nounced in other areas. e pharmaceutical interest in proteins and other microbially produced substances is steadily increasing and methods to detect and avoid endotoxins are developed and routinely used in the pharmaceutical industry (Ding and Ho, ) Furthermore, many surfactant applications require fine tuning of surfactant properties that are not easily predicted e.g. consistency, the ability to form suspensions or foam volume and stability. Basic properties such as micelliza- tion are difficult to predict even for simple mixtures of surfactants (Bergström and Eriksson, ; Khan and Marques, ; Hines, ). Many com- mercial surfactants are extremely complex mixtures that have quite different properties compared to their main components (e.g. Solutol HS and PEG mono--hydroxystearate, compared in Paper V). It is thus an advantage for the manufacturer to be able to adapt the process and the product composition to find the desired properties. is is done by varying reactants and reaction conditions to yield different product mixtures. Examples include Brij and Brij that are PEG stearyl ethers with an average of and ethylene oxide units respectively; Span and Span that are sorbitan (mono)-laurate and (mono)-oleate respectively and the APG series of surfactants where reaction temperature and time is adjusted to vary the product composition (Weuthen et al., ). Some of the advantages of enzyme catalysis in organic synthesis can be disadvantages in surfactant synthesis. e specificity of enzymatic processes and their negligible byproduct formation enables better defined products but also gives less room for process and product variation. us, enzyme ca- talysis will not replace current surfactant manufacturing processes but rather
36 37 Surfactant purity Surfactant purity
complement them for use in applications where the loss in product variability is balanced by the lower cost, environmental benefits or better performance of a well-defined product.
36 37 Selectivity
Selectivity
Generally, enzymes show a high selectivity for which molecules they accept as substrates. Many lipases accept a wide range of substrates while displaying high selectivity at the same time. is makes them useful as catalysts in chemi- cal synthesis (Gandhi et al., ; Gotor, ; Davis and Boyer, ). Chemoselectivity Chemoselectivity, or the preference for one type of chemical reaction over another, is a property so frequently encountered in enzymatic catalysis that it is sometimes forgotten. A large part of the side products in current surfac- tants are due to unspecific reactions induced by the low chemoselectivity of the manufacturing method (e.g. glycoside polymerization in acid-catalyzed esterification of sugars and transesterification or unspecific ethoxylation of hydroxyl groups in base-catalyzed ethoxylation of carboxyl groups). ese side reactions can be largely avoided by using enzymes. e well-defined active site of enzymes strictly dictates possible reaction mechanisms and the efficiency of enzymes allow for lower reaction temperatures, which decreases the number of unspecific side reactions. Efficient use of enzymatic chemoselectivity can for example be seen in Paper I and III where we used lipase to catalyze the esterification of ascorbic acid with oleic acid. Chemical synthesis of ascorbyl oleate (Hoffmann-La Roche, ; Alam and Khan, ) produces a yellow to red product because of side reactions and oxidation. Enzymatically synthesized ascorbyl oleate on the other hand, contains few byproducts and is white. Regio- and substrate selectivity Regioselectivity is the ability to discriminate between similar functional groups that are located in different parts of a molecule. In this work, regi- oselectivity is used to discriminate between primary and secondary hydroxyl
38 39 Selectivity
groups in the esterification of ascorbic acid (Paper I). Substrate selectivity is used in the esterification of PEG with -hydroxystearic acid and derivatives (Paper IV and V). e enzyme used in the present work, lipase B from Candida antarctica, prefers primary alcohols because of the geometry of its active site (Anderson et al., ). Secondary alcohols also react but with less rate enhancements. e reaction rate decreases rapidly with the distance of the hydroxyl from the end of the alkyl chain (Rotticci et al., ). e relative initial rates for esterification usingS -ethyl thiooctanoate were , , and for -octanol, -nonanol, -decanol and -undecanol respectively (Orrenius et al., ). e chemical reactivity of a primary hydroxyl group is approximately one order of magnitude higher than that of a secondary alcohol but the selectivity of the lipase greatly increases that difference. Tertiary alcohols are often so poor substrates for lipases that they can be successfully used as solvents in lipase- catalyzed esterification of primary or secondary alcohols e.g.( Paper I). In Paper I, the regioselectivity of Candida antarctica lipase B enables exclu- sive esterification of the primary -hydroxyl of ascorbic acid (Figure .). O HO O O OH HO oleic acid HO OH ascorbic acid
C. antarctica lipase B t-amyl alcohol
O HO O O O H2O ascorbyl oleate HO OH
Figure 6.1. Enzymatic synthesis of ascorbyl oleate.
In the enzymatic esterification of PEG -hydroxystearates in Paper IV the primary PEG hydroxyl groups are esterified and the secondary hydroxyl group on the fatty acid is left untouched (Figure ., R=H). In Paper V, PEG is esterified with various -acyloxystearates without reactions on the second- ary ester (Figure ., R=acyl). e lipase first reacts with the fatty acid in the acylation step, forming the acyl enzyme intermediate. Secondly, a hydroxyl group attacks the acyl enzyme in the deacylation step and the product ester is formed. In the esterification of ascorbic acid, primary and secondary hydroxyl groups on ascorbic acid compete with each other.
38 39 Selectivity Selectivity
O H R2 O O HO n O R1 ethyl 12-hydroxystearate PEG
C. antarctica lipase B
O H O O HO R2 O n R1 PEG 12-hydroxystearate water or ethanol (removed by vacuum)
Figure 6.2. Enzymatic synthesis of PEG 12-hydroxystearate. (R1=H) and PEG 12-acyloxy-stearates (R1=C2-C18 acyl). In Paper IV, free acid (R2=H) was used. In Paper V, ethyl esters (R2=C2H5) were used.
In the PEG ester syntheses, the primary hydroxyl groups on PEG compete with the secondary hydroxyl group on -hydroxystearic acid. In both of these cases, the regioselectivity of the reaction is thus introduced in the deacyla- tion of the enzyme. Substrate competition in the PEG -hydroxystearate synthesis is illustrated in Figure ..
OH OH H2O 5 9 O 12-hydroxystearic acid Selection OH HO Enzyme O Enzyme 5 9 O OH Acyl enzyme O H HO H O O 5 9 O n n PEG 12-hydroxystearate PEG (product)
O OH O R 5 9 O 5 9 OH O O R 5 9 12-hydroxystearic acid or O PEG 12-hydroxystearate
Figure 6.3. Competition between primary and secondary hydroxyl groups in enzymatic synthesis of PEG 12-hydroxystearate. Hydroxyl groups determining the selectivity are marked in bold.
40 41 Selectivity Selectivity
e reactions are run for extended times and high selectivity for primary hydroxyl groups or esters is required to produce pure products. With equi- molar amounts of reactants, the concentration of secondary hydroxyl groups competing for deacylation (Figure ., lower path) is comparable to the concentration of primary hydroxyl groups (Figure ., middle path). Still, no secondary esters must form. In Paper IV, an excess of PEG is used to decrease the amount of PEG diesters but it also increases the chance of a primary hydroxyl being used as a substrate. In the esterifications described in Paper V, substrate competition occurs already at the acylation of the enzyme as there are two carbonyl groups avail- able in the -acyloxy-stearates. e desired reaction is acylation of the en- zyme with the carbonyl group of the -hydroxystearic acid (Figure ., lower path). e carbonyl groups in the acyloxy moieties (R) in both substrate and product may acylate the enzyme as well (Figure ., upper path). e two acyl groups are present in equal amounts in the -acyloxy stearate throughout the reaction and as the reaction runs for extended times to reach completion, regioselectivity becomes very important to avoid byproduct formation. If the acyloxy ester was accepted as acyl donor by the enzyme in only a fraction of the reactions, the product mixture would include PEG esters of the acyloxy acid (R), PEG esters of -hydroxystearic acid and PEG esters of oligomers of -hydroxystearic acid (estolides). O R O 1 O 5 9 R OH O 1 5 9 R O R2 O 2 Enzyme
O O 12-acyloxy-stearic acid or PEG 12-acyloxy-stearate R 2 O OH 5 9 O H2O Selection 12-acyloxy- R2 stearic acid O HO Enzyme O Enzyme O 5 9 O R O 2 Acyl enzyme O H HO H O O 5 9 n n O PEG 12-acyloxystearate PEG (product) Figure 6.4. Competition between acyl groups in enzymatic synthesis of PEG 12-hydroxystearate. Hydroxyl groups determining the selectivity are marked in bold.
40 41 Selectivity Selectivity
A large excess of PEG is used in the reactions and the product mixture would in the case of insufficient regioselectivity approach almost total alco- holysis of the acyloxy stearate forming primary PEG esters and thus not yield the pure products now presented. Stereoselectivity Most chiral natural materials used in surfactant synthesis (e.g. sugars, amino acids, hydroxy fatty acids) are naturally enantiomerically pure. Accordingly, most natural surfactants are also enantiomerically pure. Enantiomerically pure surfactants display some special properties that are used only in a very limited number of applications such as the synthesis (Doiuchi and Minoura, ; Ohkubo et al., ; Fehring et al., ) and separation (El Rassi, ; Otsuka and Terabe, ; Yarabe et al., ) of chiral compounds. Current chiral surfactants used in these applications are either natural, such as lecithin (Doiuchi and Minoura, ) or produced from chiral reactants such as amino acids (Diego-Castro et al., ), glycosides (Fehring et al., ) or other compounds (Fornasier and Tonellato, ; Ot- suka and Terabe, ) and stereoselectivity is rarely used in their synthesis. PEG monoesters PEG monoesters are better solubilizers than PEG diesters. In order to obtain the PEG monoesters, only one end of each PEG chain must be acylated. is is not a matter of selectivity as the two hydroxyls in the PEG chain are chemically equivalent and cannot be distinguished from each other by the enzyme. e simplest way to ensure predominant formation of monoester is to use an excess of PEG (Hilditch and Rigg, ). is unfortunately leaves a large excess of free PEG in the product that has to be removed. PEG monoalkyl ethers To completely avoid formation of PEG diesters, PEG can be replaced by a PEG alkyl ether such as PEG monomethyl ether (MePEG). is levaes only one free hydroxyl group available for esterification and MePEG do not need to be in excess. We have performed several enzymatic esterifications using only a slight excess of MePEG (. times the fatty acid) to avoid free acid in the product and we noted that the reactions worked well but were much slower than corresponding PEG esterifications using ten times PEG excess (unpublished data). At the end of the PEG and MePEG esterifications there is only a small amount of free fatty acid (or its ethyl ester in some of the reactions) left to compete with large amounts of PEG ester for the acylation of the enzyme.
42 43 Selectivity Selectivity
Net product formation first requires the free acid (or its ethyl ester) to acylate the enzyme and this step is comparable for the PEG and MePEG syntheses. Secondly, a free hydroxyl group must attack the acyl enzyme to form the product. At the end of the reaction there is relatively seen only . equivalents of hydroxyl groups available in the MePEG reaction compared to equiva- lents still available in the PEG reaction (based on . and times excess of MePEG and PEG respectively). is difference may well account for the ap- proximately - to -fold longer reaction times needed when using MePEG in . times excess compared to PEG in times excess (unpublished data). Apart from the advantage of exclusive monoester formation when using MePEG, esters of MePEG cannot spontaneously rearrange to diesters upon storage, as has been reported for PEG monoesters (Parris and Weil, ). By exchanging the free hydroxyl group of PEG to the methyl ether in MePEG, the properties of the hydrophilic part of the surfactant is slightly altered. e change is usually considered equivalent of removing three EO units from the PEG chain and thus has a relatively small effect on the surface- chemical properties (Conroy et al., ). Borate esters Another synthetic route to PEG monoesters uses borate intermediates (Fig- ure .). PEG is first reacted with boric acid in ratio : resulting in a mixture of free PEG and PEG borates. e PEG borates can then be esterified in the free PEG end. e borate esters are selectively hydrolyzed in water and the main product is PEG monoester (Hartman, ; Gerhardt and Holzbauer, ; Rao et al., ). We performed initial trials combining enzymatic esterification and PEG borates and found only negligible formation of PEG ester using Candida antarctica lipase B (unpublished results). Boric acid and borates are known to be efficient inhibitors for many proteases (von Euler and Kertecz, ). ey bind covalently to the active site serine and form a tetrahedral transi- tion state analog that inhibits the enzyme (Antonov et al., ; Bauer and Pettersson, ). Lipases and proteases are serine hydrolases and have similar mechanisms and inhibition of the lipase by borate esters may be the reason for the low productivity. H O O H OH 100 ºC, vacuum O H O B n 3 HO HO B O O H n OH n O n PEG boric acid tri-PEG borate Figure 6.5. Synthesis of PEG borate.
42 43 Selectivity
Ethoxylation of fatty acids PEG esters have been used as surfactants for a long time (Goldschmidt, ; Kosswig, ) and are synthesized industrially via base-catalyzed eth- oxylation of fatty acids (Stockburger, ). e process yields a well-defined pattern of monoester, diester and PEG formation. Ethoxylation of oleic acid is briefly presented here for a comparison with the enzymatic methods. e reaction starts with the ethoxylation of the free oleic acid via several reactions that includes fast transfer of the acidic proton of oleic acid to the alkoxide ion formed by addition of ethylene oxide to the acid. Ethoxylation of the oleate dominates and after the addition of one equivalent of ethylene oxide (EO) the ratio of monoester to diester is .:. As the ethoxylation continues, no oleic acid is available for proton transfer and the dominating reaction becomes elongation of the PEG chain. Sodium hydroxide is used as polymerization catalyst but also catalyzes transesterifi- cation and the ratios of PEG, monoester and diester rapidly approach equi- librium. When . EO equivalents have reacted, the monoester to diester ratio has dropped to .: and the molar ratio of PEG, monoester and diester only changes slowly with further EO addition and stabilizes at approximately .: :. e theoretical ratios, if all hydroxyls are equally reactive, are :: but water in the starting materials increases the PEG content (Stockburger, ). e ratio of monoester to diester varies with the reaction conditions and may approach values as low as : (Wrigley et al., ). If -hydroxystearic acid is used as starting material, the sodium hydroxide catalyzes ethoxylation as well as acyl migration on the -hydroxyl group yielding a complex mixture of PEG mono- and diesters of -hydroxystearic acid and oligomers thereof, as well as ethoxylated -hydroxyl groups terminated with free hydroxyl groups and a variety of esters.
44 45 Selectivity
Environmental aspects
With increased public awareness and concerns of global warming, the chemical industry and the rest of society strive to decrease the use of limited resources and to increase the use of sustainable technologies. To improve sustainability and decrease the environmental impact of industrial chemical production we need to use renewable raw materials instead of fossil materials and fuels (Anastas and Kirchhoff, ). e present work was performed within the framework of the Competence Centre for Surfactants based on Natural Products, SNAP (SNAP, ). e aims of SNAP include the design, synthesis and characterization of new sur- factants with improved function and less environmental impact than present alternatives. Natural products and biocatalysis As the importance of environmentally friendly processes increases, so does the economic value of using and producing natural products. e definition of “natural” varies between different sources but usually include materials isolated from vegetable or animal sources that may be modified with microbiological, enzymatic or mild chemical processes. is definition holds for regulations in EU and USA. For example, a natural flavoring substance is defined as: “[Natural flavoring substance are] obtained by appropriate physical pro- cesses (including distillation and solvent extraction) or enzymatic or microbio- logical processes from material of vegetable or animal origin either in the raw state or after processing for human consumption by traditional food-prepara- tion processes (including drying, torrefaction and fermentation)” (EU, )
44 45 Environmental aspects Environmental aspects
“e term natural flavor or natural flavoring means the essential oil, oleo- resin, essence or extractive, protein hydrolysate, distillate, or any product of roasting, heating or enzymolysis, which contains the flavoring constituents derived from a spice, fruit or fruit juice, vegetable or vegetable juice, edible yeast, herb, bark, bud, root, leaf or similar plant material, meat, seafood, poultry, eggs, dairy products, or fermentation products thereof, whose significant func- tion in food is flavoring rather than nutritional.” (FDA, ) In this context, enzymatic synthesis can add considerable value to products. A striking example is vanillin, the compound responsible for the characteristic flavor of vanilla. True vanillin is extracted from the beans of the orchid Vanilla planifolia with a worldwide production of approximately tons per year. is consti- tutes less than . of the total vanilla market of , tons per year. e remaining . of vanilla flavor is synthetic and is mainly produced from eugenol, guaiacol or lignin-containing waste from paper industry. Vanillin can also be produced from the same precursors by fermentation and enzymatic methods and is then considered natural (Rao and Ravishankar, ). e market price for true vanilla was approximately . USD/kg in . At the same time, vanilla flavor synthesized with biocatalysts had an estimated production cost of . USD/kg and a market price of around . USD/kg (Feron et al., ). is should be compared to the market price of USD/kg for chemically synthesized vanillin (Krings and Berger, ). Clearly, the economical value of a having a natural product can be an important factor for using enzymatic synthesis. Natural surfactants e term “natural surfactants” usually describes surfactants produced only from natural materials under mild conditions (e.g. glycerides, sugar esters and peptide surfactants) (Holmberg, ). e term “natural surfactants” sometimes also describes surfactants produced with raw materials that could be natural materials but are in fact produced from fossil materials or using harsh chemical processes. Approximately (. million tons/year) of the global fatty alcohol production is used in detergents. Only - of the world production of fatty alcohols is of vegetable origin and is produced by catalytic hydrogenation of fatty acid methyl esters at severe conditions, i.e. °C and bar. e remaining of fatty alcohols is of petrochemical origin (Tsushima, ). In this study, we used natural products (ascorbic acid and various fatty acids) for the synthesis of surfactants as well as polyethylene glycol (PEG) which is produced from fossil materials. Other hydrophilic groups (e.g. car-
46 47 Environmental aspects Environmental aspects
bohydrates) are increasingly replacing PEG in many applications. Pharma- ceutical solubilization is one area where PEG surfactants are hard to replace (Söderlind et al., ). In this work, we have improved the synthesis of PEG esters, yielding better defined surfactants with enhanced properties (Paper IV and V). To replace PEG surfactants in pharmaceutical solubilization it is important to understand the mechanisms behind their behavior. Well-defined surfactants are important for structure-function studies. Biological surfactants Surfactants directly isolated from organisms with no further derivatization are often called biological surfactants. Biological surfactants may be isolated from crops (e.g. lecithin from soy beans) or produced via microbial fermentation (e.g. glycolipids such as sophorose lipids produced by Candida bombicola or rhamnolipids produced by Pseudomonas aeruginosa) (Garti, ; Lang, ). Biological surfactants also include protein and carbohydrate amphiphiles but these are not used on a large scale (Garti, ). Natural raw materials Many of the natural hydrophobic groups used to produce surfactants (e.g. fatty acids, sterols) are isolated from complex sources and are available in larger quantities only in technical grades because of the high costs of purifi- cation. For example, several interesting fatty acids (e.g. oleic acid, erucic acid, ricinoleic acid) are sold in - purity. Similar purity problems occur to a lesser extent for the hydrophilic groups. Polymeric carbohydrates (e.g. dextrin and dextran) often have very wide distributions in molecular weight and/or a high content of monomeric sugars. To make new environmentally friendly surfactants for a wider range of ap- plications, researchers need to broaden the range of starting materials (Cheva- lier, ). Some of the best current surfactants (e.g. nonylphenol derivatives) have severe environmental impact and it is important to find alternatives. e size of the hydrophilic group determines many properties of the sur- factant. Available carbohydrate raw materials are monomeric (e.g. glucose, fructose and sorbitol), dimeric (e.g. sucrose and lactose) or polymeric (e.g. starch, pectin, chitin, seaweed polysaccharides and dextran). However, only few natural oligomeric sugars are available and commercial surfactants are often synthesized by polymerization of monomeric sugars (von Rybinski and Hill, ). One alternative is to produce oligomeric carbohydrates using enzymes, for example by transglycosylation of fructooligosaccharides (Sheu et al., ; Röper, ) or by hydrolysis of xyloglucan to well-defined oligosaccharides
46 47 Environmental aspects Environmental aspects
(Marry et al., ). Carbohydrates can also be processed to a variety of build- ing blocks useful for surfactants and other chemical synthesis (Reintjes and Cooper, ; Lichtenthaler and Mondel, ). Natural fatty acids are widely used in surfactants. Approximately . million tons/year () of the global fatty acid production were used in detergents in (Tsushima, ). However, few fatty acids dominate the market. Un- common fatty acids (e.g. acetylenic, saturated, unsaturated epoxy, conjugated and non-conjugated polyolefinic) are not commercially available even though they occur in plants, animals and marine organisms (Spitzer, ; Johansson and Svensson, ; Leray, ). ese fatty acids as well as derivatives of traditional fatty acids present possibilities to use other functionalities than the carboxylic acids for surfactant synthesis (Biermann et al., ; Hedman et al., ). Genetically modified organisms Genetically modified organisms may simplify the production of rare or even unnatural substances including carbohydrates and fatty acids (Lee et al., ; Dunwell, ; Murphy, ). Genetically engineered crops are already widespread. As much as of the soybean and of cotton acreage in USA used pesticide resistant variants in . In the same year, insect protection with genetically introduced Bt toxin was employed in of corn and of cotton acreage (Fernandez-Cornejo and McBride, ). e increased use of genetically modified organisms (GMO) has immense economical, political, ethical, scientific and environmental impact. Whether products isolated from GMO are natural products, if large scale use of GMO is safe, and if products from GMO will (and should) be accepted by the con- sumers is under fierce debate. Recent events under discussion are the migra- tion of genes in genetically modified corn to wild and cultivated populations (Quist and Chapela, ; Butler, ; Christou, ; Quist and Chapela, ; Salleh, ); transgenic corn with pesticidal Bt toxin and its effect on butterflies (Hodgson, ; Losey et al., ; Niiler, ; Hansen Jesse and Obrycki, ; Hodgson, ; Wraight et al., ) and the fact that geneti- cally modified Bt-corn that was not approved for food use nevertheless found its way to American and Japanese food suppliers (Uchtmann, ). For genetically modified crops, as for other natural products, much of the economic benefits depend on public acceptance and on marketing advantages. e future of GMO in chemical, insecticidal and pharmaceutical applications remains to be seen.
48 49 Environmental aspects Environmental aspects
Natural and healthy Using natural products and enzymatic synthesis in general gives compounds that are easily degraded in biological systems. However, the isolation of natu- ral raw materials may cause problems. For example, -hydroxystearic acid used in Paper IV and V is produced after hydrogenation of oil from the seeds from the castor plant (Ricinus com- munis). Castor seeds contain a glycoprotein that is one of the most allergenic compounds known and practically all persons exposed to powdered castor beans develop serious allergic reactions (orpe et al., ; Kemeny, ). e seeds also contain ricin, a protein that is one of the most toxic substances known (McKeon et al., ). USA, a major consumer of castor oil and once a major producer, today has no production mainly because of occupational health problems among the workers. e annual production of castor seed is approximately ,, tons from which oil can be extracted. e largest producer is India () followed by Brazil () (Adya Oils, ). Castor oil is mainly used for polymers, paints and lubricants and is classified as strategically important in many countries. Much work has been done to find and use similar oils from other crops (Carlson et al., ; Hayes and Kleiman, ; Hayes et al., ) and to reduce the toxicity of castor plants by genetic modification (McKeon et al., ). Other aspects Factors beyond the raw materials also determine the environmental impact of a chemical process (Anastas and Kirchhoff, ). e subject is beyond the scope of this work but some aspects will be mentioned. e use of organic solvents should be minimized for both economic and environmental reasons. e importance of solvent-free processes (Paper IV and V) and new alternatives such as ionic liquids (Paper II and III) will in- crease. If the use of solvents cannot be completely avoided it should be limited to non-toxic options. To lower the cost by reusing enzymes, they must not be inactivated by the physical and chemical reaction conditions. e most stable enzymes generally come from organisms that grow under extreme conditions (e.g. temperature, pH, salt concentration and/or pressure) (Hough and Danson, ; Demirjian et al., ; Schiraldi and De Rosa, ). However, the lipases used in most chemical synthesis come from mesophilic organisms and tolerate surprisingly harsh conditions compared to their natural environment at temperatures of - °C. Immobilization of an enzyme further improves the thermal and
48 49 Environmental aspects chemical stability and facilitates handling of the catalyst and its separation from the reaction mixture (Klibanov, ; Tischer and Kasche, ). e enzyme used in the present work, lipase B from Candida antarctica, is active at pH .-. with its optimum at pH (Anderson et al., ). In aqueous buffers, its activity does not degrade up to approximately - °C (Anderson et al., ; Arroyo et al., ). It is stabilized in dry conditions and can successfully be used at - °C in organic media for extended times (Cao et al., ) and Paper IV. Compared to chemical catalysts, enzymes are relatively sensitive. Never- theless, their industrial use is steadily increasing and already includes several large-scale processes as for example enantiopure alcohols, acrylamide, aspar- tame and penicillins (Schmid et al., ). eir biological origin also gives them environmentally favorable properties such as biodegradability.
50 51 Environmental aspects
Functional studies
Ascorbyl esters Ascorbic acid, or vitamin C (Figure .), was the first vitamin to be discovered. In the Scottish physician James Lind found the cure for scurvy in one of the first modern medical experiments (Lind, ). He divided a group of British navy sailors with scurvy into six groups, gave them different treat- ments and found that those who ate two oranges and a lemon per day were cured in only six days. e Royal Navy started to bring lemon juice on their ships almost years later – a delay that cost the lives of many thousands of sailors (Lloyd, ). HO HO HO O O O HO O oxidation HO O + H2O O reduction O OH HO OH O O OH OH ascorbic acid dehydroascorbic acid dehydroascorbic acid (hydrated hemiketal)
Figure 8.1. Ascorbic acid and its oxidation product, dehydroascorbic acid. The first and second pKa of ascorbic acid is approximately 4.1 and 11.8 for the protons on the 3-hydroxyl and 2-hydroxyl, respectively.
Almost years after the first discovery, vitamins were again brought into scientific focus (Hopkins, ) and were named from vital amines (Funk, ). “Hexuronic acid” was purified and isolated (Szent-Györgyi, ) and found to be identical to vitamin C (Svirbely and Szent-Györgyi, a; b). It was named ascorbic acid from anti-scorbutic when the initial suggestions ignose and godnose was refused by the journal editor (Szent-Györgyi and Haworth, ; Szent-Györgyi, ). It was also the first vitamin to have its structure determined (Herbert et al., ) and a synthetic route was soon devised (Ault et al., ). For their work on vitamins, Hopkins received the
50 51 Functional studies Functional studies
Nobel Prize in physiology or medicine in , Szent-Györgyi in and Haworth received it the same year in chemistry. Ascorbic acid is today produced in approximately . tons/year, mainly using the Reichstein process that was devised only a few years after the dis- covery of ascorbic acid (Reichstein et al., ; Lichtenthaler and Mondel, ). Biotechnological approaches for intermediate steps in the process have been employed since the earliest days, for example in the microbial oxidation of -sorbitol to -sorbose (Reichstein and Grussner, ). e technology has developed over the years and some recent processes are purely microbial (Hancock and Viola, ). O HO O O O
HO OH ascorbyl palmitate O HO O O O
HO OH ascorbyl oleate O HO O O O
HO OH ascorbyl erucate Figure 8.2. Ascorbyl fatty acid esters.
Ascorbic acid is used as an antioxidant in foods and cosmetics. It is not soluble in fats and oils where antioxidants are needed to preserve polyunsatu- rated fatty acids or other components with high nutritional value. Already a few years after the discovery of ascorbic acid, ascorbyl palmitate (Figure .) was synthesized for use as an antioxidant in fats and oils (Hoffmann-La Roche, ; Swern et al., ). Ascorbyl palmitate is still extensively used but is crystalline and not very soluble in oils, which limits its applications. e balance between crystal lattice energy and solvation energy deter- mines the solubility of a compound. Solvation energies are similar for similar compounds and the melting point is a measure of the crystal lattice energy (Woudenberg-van Oosterom et al., ). us, the solubility of similar com- pounds is inversely proportional to their melting points. e crystallinity of a
52 53 Functional studies Functional studies
compound can be estimated more precisely with its enthalpy of fusion, which is the enthalpy change needed to melt the compound. In Paper I, we exchanged the palmitic acid to oleic acid and introduced an irregularity (i.e. a double bond) in the alkyl chain which decreased the crystal- linity. e melting point of ascorbyl oleate is accordingly much lower than for similar saturated esters (Figure .). e enthalpy of fusion decreased four- to five-fold compared to ascorbyl palmitate (Table .). 150 Tanaka (1966) Palma (2002 & 2003) 140 Swern (1943) This study 130
120
110
100 Melting point (°C) 90
80
2 4 6 8 10 12 14 16 18 20 22 Number of carbons in acyl chain
Figure 8.3. Melting points of ascorbyl fatty acid esters. All esters are saturated except ascorbyl oleate (C18:1) from Paper I and ascorbyl erucate (C22:1, unpublished).
Using the same methods as for ascorbyl oleate, we synthesized ascorbyl eru- cate (not published). Ascorbyl erucate was found to be polymorphic, having several crystal structures, with phase transitions at - °C and °C. Further studies were done only with ascorbyl oleate due to its lower crystallinity. Table 8.1. Physical properties of ascorbyl fatty acid esters.
Melting point Enthalpy of fusion Behavior (°C) (J/g) Ascorbyl palmitate Crystalline 114-120 129 Ascorbyl oleate Amorphous 83-84 20-30 Ascorbyl erucate Not measured Polymorphic 88 Antioxidative effect of unsaturated ascorbyl esters To determine the efficiency of ascorbyl oleate as a fat-soluble antioxidant, we measured the peroxide formation and oxidation of tocopherols in rapeseed oil when stored. Oxidation of rapeseed oil gives a rancid taste. Consumers
52 53 Functional studies Functional studies detect rancidity at peroxide values of about - milliequivalents/kg (meq/ kg) and higher (Malcolmson et al., ). Tocopherols are naturally present antioxidants in the oils, belong to the group of vitamin E compounds and are consumed in the oxidation processes. Ascorbyl oleate is a more effective antioxidant than ascorbyl palmitate in rapeseed oil (Paper I). For rapeseed oil without addition of antioxidant, a peroxide value of meq/kg was reached within weeks at °C. With the addition of . g ascorbyl palmitate/kg oil, the value was not reached until after weeks. Using the same concentration of ascorbyl oleate, the time re- quired increased to almost weeks (Paper I, Figure ). After weeks at °C the unprotected oil had lost all its tocopherols, the ascorbyl palmitate sample had less than γ-tocopherol left and the oil protected by ascorbyl oleate had retained and of γ- and δ-tocopherols respectively. Our results differ from other evaluations where similar antioxidant activities have been found for free ascorbic acid, ascorbyl palmitate and the unsaturated ascorbyl linoleate (Watanabe et al., ; Watanabe et al., ). Watanabe et al. studied the oxidation of polyunsaturated fatty acids (Figure .) and their ascorbyl esters at high temperature ( °C), short oxidation times (- hours) and with high antioxidant concentrations (-). To reach significant protection, high concentrations of antioxidant were required (- mol-, or - g/kg). O
HO linoleic acid O
HO α-linolenic acid O
HO γ-linolenic acid O
HO arachidonic acid O
HO docosahexaenoic acid Figure 8.4. Polyunsaturated fatty acids used in Watanabe et al. (2001).
In our study, we followed the oxidation of low-erucic acid rapeseed oil (Figure .) which mainly consists of triglycerides of unsaturated fatty acids. We also used lower temperatures (- °C), longer times (- weeks) and low antioxidant concentrations ( mg/kg). e results indicated that the use of ascorbyl oleate instead of ascorbyl palmitate could increase the shelf-life of food oils.
54 55 Functional studies Functional studies
O palmitic acid 4.5 % O
R stearic acid O 1.5 % O R O R = oleic acid 58.5 % O O R linoleic acid 21.5 % O α-linolenic acid 10.5 % Figure 8.5. Composition of rapeseed oil used in Paper I.
ere are other examples where vitamins and similar compounds have been enzymatically esterified to improve their properties. One example is bixin, a carotenoid, that was esterified with ascorbic acid to protect it from oxidation (Humeau et al., ). Another example is the derivatization of retinol (vita- min A) to the diester retinyl sorbityl adipate, increasing its aqueous solubility (Rejasse et al., ). Pharmaceutical solubilization Many of the active substances used in drugs are hydrophobic but must be administered as aqueous preparations (Figure .). is can be done using surfactants that form micelles in which the active substance is contained. Surfactants used to improve the solubility of other compounds are called solubilizers. Pharmaceutical solubilizers must not cause negative effects when administered to a patient. In some applications (e.g. injection in the blood stream) this is extra important. Cl
Cl O O O O
N H Felodipine Figure 8.6. The water-insoluble drug that was used in the solubilization studies.
54 55 Functional studies Functional studies
Surfactant effects on cells Most surfactants show strong disruptive effects on cells and cell membranes (Helenius and Simons, ; Jones, ). is is significant e.g. in the injec- tion of a solubilized drug when blood cells are subjected to relatively high concentrations of surfactant. e mechanisms of surfactant-induced cell lysis are not fully understood, partly because of the extremely complex structure of cell membranes. Cell membranes are often described as lipid bilayers but they only contain - lipids. e remainder is made up of proteins (-) and some oligosaccha- rides (). Cell membranes are highly unsymmetrical and some lipids are found mainly on the inside and others mainly on the outside of the membrane. Proteins are immersed in the membrane to various degrees, are oriented to- wards a specific side of the membrane or may flip to change orientation. Cell membranes are also laterally heterogeneous, with an uneven distribution of lipids and proteins over the cell surface (Kondo, ; Jones, ). Already at very low concentrations surfactant molecules integrate with the cell membrane and may stabilize the cells against lysis (Reber, ; Trägner and Csordas, ). As the surfactant concentration increases, the cell membrane is destabilized and its ion permeability increases. Osmosis causes swelling of the cell and may finally result in lysis (Trägner and Csordas, ; Bielawski ). Surfactants also cause considerable redistribution of membrane components and extraction of lipids and proteins already at low, non-lytic concentrations. e specific effects and the distribution of extracted materials depend on the type of surfactant (Maher and Singer, ). At suf- ficiently high concentrations, the cell membrane is completely solubilized ( Jones, ). Hemolysis caused by solubilization often occurs around the critical micelle concentration (CMC) of the surfactant and is a relatively fast process com- pared to osmotic hemolysis (Shalel et al., ). e mechanisms of hemolysis imply that efficient solubilizers are often also powerful hemolytic agents. e effect of surfactants on cells can also be measured with transepithelial electrical resistance (TEER) which is the resistance across a layer of epithelial cells. We used a monolayer of human intestinal cells (Caco-) cultivated on a membrane. Surfactants are well known to affect the proteins connecting the cells. Disruption of the cell layer strongly affects inter-cell connections and lowers the electrical resistance over the membrane. At moderate levels, the disturbances are reversible. At higher levels of disruption, cell damages become permanent (Dimitrijevic et al., ; Ward et al., ; Paper V). Oxidation of PEG results in the formation of aldehydes (Decker and Marchal, ) which irritates the skin and causes allergic reactions (Bergh et al., ). Peroxides formed in the oxidation of PEG surfactants can also
56 57 Functional studies Functional studies
induce hemolysis but free PEG does not display the same hemolytic effect even though the oxidation processes are similar (Azaz et al., ; Segal and Milo-Goldzweig, ). is could be explained by that incorporation of sur- factant into the cell membrane brings the release of peroxides closer to the cell, increasing the risk of cell damage. Peroxide hemolysis was inhibited by addition of antioxidants. Possibly, ascorbyl fatty acid esters could be added to the solubilizer in order to decrease peroxide dependent hemolysis. PEG-esters of hydroxy fatty acids Many ethoxylated fatty acids and oils are surfactants with very favorable hemolysis behavior despite that they are efficient solubilizers. Commercial products include ethoxylated -hydroxystearic acid (Solutol HS , Figure .); ethoxylated castor oil (Cremophor EL, Figure .) and ethoxylated sor- bitan monooleate (Tween ). Commercial samples of these surfactants are very complex mixtures and often have large batch-to-batch variation. H or O R1 O R2 O R3 R1 O R2 = O n1 n2 R3 or m O
10
8 PEG, 53% PEG mono-(12-hydroxy stearate), 23% PEG di-(12-hydroxy stearate), 10%
y (%) y PEG tri-(12-hydroxy stearate), 7% it 6 PEG tetra-(12-hydroxy stearate), 4% ens
t PEG penta-(12-hydroxy stearate), 2% in PEG hexa-(12-hydroxy stearate), 1%
ve ve 4 ti la Re 2
0 500 1000 1500 2000 2500 3000 m/z
Figure 8.7. Components in ethoxylated 12-hydroxystearic acid and the mass spectrum of Solutol HS 15. Technical grade 12-hydroxystearic acid contains 10- 20% stearic acid. In Solutol HS 15, one mol of 12-hydroxystearic acid is reacted with 15 mol of ethylene oxide resulting in n1+n2=15; m is usually below 3. The MALDI-TOF spectrum is based on one measurement and intensities may not be proportional for different compounds.
56 57 Functional studies Functional studies
O R1 O
n1 R1 O R R2 R4 O 2 or O O n O R3 O 4 n2 m R = O 4 R3 or O R5 n3 O R6 or H O R6 R5 O n5
10 PEG, 29% PEG mono-ricinoleate, 16% PEG di-ricinoleate, 10% 8 Glyceryl-PEG, 19% Glyceryl-PEG mono-ricinoleate, 14% y (%) y
it Glyceryl-PEG di-ricinoleate, 8% 6 ns Glyceryl-PEG tri-ricinoleate, 3% te Glyceryl-PEG tetra-ricinoleate, 1% in
ve ve 4 ti la Re 2
0 500 1000 1500 2000 2500 3000 m/z
Figure 8.8. Components in ethoxylated castor oil and the mass spectrum of Cremophor EL. Fatty acid composition is 90% ricinoleic acid (12-hydroxyoleic acid), 4% linoleic acid, 3% oleic acid and small amounts of stearic, palmitic acid and dihydroxystearic acid. In Cremophor EL, one mol of castor oil is reacted with 35 mol of ethylene oxide, resulting in n1+n2+n3+n4+n5=35; m is unknown but probably below 2-3. The MALDI-TOF spectrum is based on one measurement and intensities may not be proportional for different compounds.
To find well-defined surfactants that have as favorable properties as the complex mixtures, we synthesized surfactants selected from the main com- ponents of commercial products. ese included PEG mono- and diesters of stearic acid and -hydroxystearic acid (Figure . and Paper IV). O O R1 O n
O O R2 O n OH
Figure 8.9. Mono- and diesters of PEG with stearic acid and 12-hydroxystearic acid. R1 and R2 is H for monoesters and stearoyl or 12-hydroxystearoyl for diesters.
58 59 Functional studies Functional studies
Surface-chemical characterization showed that the PEG diesters were to hydrophobic and had too low solubility to be practically used as solubilizers. PEG monostearate induced hemolysis and affected TEER at low concen- trations (data not shown). ese results are consistent with other reports on ethoxylated stearic acid (Ohnishi and Sagitani, ). Ethoxylated - hydroxystearic acid (Solutol HS ) is a surfactant with very small effects on hemolysis and TEER and its main component is often stated to be PEG mono--hydroxystearate. However, our tests showed that pure PEG mono--hydroxystearate had severe effects and was not a good pharmaceu- tical solubilizer. PEG 12-acyloxy-stearates Large hydrophilic and hydrophobic groups often renders a surfactant less hemolytic because it decreases its negative effects on the cell membranes (Kondo, ; Miyajima et al., ; Ohnishi and Sagitani, ; Reinhart and Bauer, ; Söderlind et al., ). Based on PEG -hydroxystearate, the size of the hydrophobic group increased by esterification of the hydroxyl group on the -hydroxystearic acid. To maintain the solubility of surfactants with large hydrophobic groups, the size of the hydrophilic group was also increased. e resulting surfactants are described in Figure . and were obtained in high purity. e only impurities being small amounts of diesters. Names and abbreviations are defined inPaper V, Table I. e PEG-CC surfactant showed improved hemolytic behavior compared to the PEG surfactants with shorter chains. PEG-CC and longer were too insoluble in water to be further studied. PEG-CC induced hemolysis above mM. PEG-CC and longer surfactants proved to be very efficient solubilizers and displayed no effect on hemolysis up to the maximum measured concentrations of mM ( g/l). For transepithelial electrical resistance (TEER), similar effects were found. PEG-CC was considerably more tolerable for the Caco- cells than its shorter analogs and showed no effects up to the maximum measured con- centration of mM. Using PEG-CC, no effects on TEER was seen below mM and PEG-CC and longer displayed no effects even at concentrations as high as mM ( g/l). e surfactants PEG-CC and shorter were good solubilizers whereas PEG-CC and PEG-CC showed lower solubilization capaci- ties. PEG-CC also showed signs of phase separation around - mM. e low solubilization capacities and solubility indicate that micelle forma- tion may be unfavorable. e absence of micelles with PEG-CC is the probable reason for its low effects on hemolysis and TEER.
58 59 Functional studies Functional studies
O
O HO O O n O
10 PEG1500 mono-(12-myristoyloxy stearate), 98%
8 PEG1500 di-(12-myristoyloxy-stearate), 2% (%) y y
it 6 ens t in 4 ve ve ti la Re 2
0 1000 1500 2000 2500 3000 m/z
Figure 8.10. Structure of polyethylene glycol (1500) mono-12-myristoyloxy- stearate (PEG1500-C18C14) and its mass spectrum. The MALDI-TOF spectrum is based on one measurement and intensities may not be proportional for different compounds.
PEG-CC and longer surfactants showed no effect on hemolysis and TEER even at high concentrations and were excellent solubilizers. is behavior has not been observed for other pure PEG-based surfactants and the surfactants are expected to be useful as pharmaceutical solubilizers for e.g. intravenous injections. Structure vs. function ere have been many attempts to correlate the hemolytic activity of a surfac- tant with its structure and physical properties. For surfactants based on PEG, poor or no correlation to hemolytic power has been found for hydrophilic- lipophilic balance (Zaslavskii et al., a) or critical micelle concentration (Zaslavskii et al., b). In general it seems that the size and structure of both the hydrophobic and the hydrophilic group determine the hemolytic activity of the surfactant (Miyajima et al., ; Ohnishi and Sagitani, ). e general shape of a surfactant molecule is determined by the size of its hydrophilic head group (with coordinated water) in relation to its hydro- phobic tail. e shape of the individual molecule determines the curvature of surfactant layers, which is important in interaction with micelles and mem- branes. Ohnishi and Sagitani recommended using bulky hydrophobic groups (three or four fatty acids) because those changed the membrane curvature less than single-chain surfactants (Ohnishi and Sagitani, ). In our study,
60 61 Functional studies Functional studies
the difference in structure, bulkiness, CMC, hydrophilic-lipophilic balance and solubilizing capacity are small when comparing PEG-CC and PEG-CC. Nevertheless, they display quite different hemolysis and TEER properties (Paper V). Future studies e major drawback of current low-hemolytic solubilizers is that they contain components that induce release of histamine, causing severe reactions such as anaphylactic shock, when injected in the blood stream. e behavior is seen with ethoxylated castor oils (Lorenz et al., ) but has also been found with commercial and purified preparations of various PEG -hydroxystearates (Zirnstein et al., ). Commercial solubilizers and their components have also been shown to be cytotoxic, having effects on drug transport and pharmacokinetics as well as extracting softeners from polyvinyl chloride (PVC) bags and tubing used for drug solutions (Gelderblom et al., ; Singla et al., ). To understand the behavior of PEG -acyloxy-stearates and to use their full potential, further pharmacological and surface chemical evaluations are needed. PEG -acyloxy-stearates can be produced in pure qualities and it is easy to vary their structure. is will help to increase our understanding of the many biological effects of surfactants.
60 61 Acknowledgements
Acknowledgements
is thesis would not have been possible without the expertise, support and inspiration I have received from many persons. First of all, I would like to thank my supervisor Professor Karl Hult for being the guide on this scientific journey. His overview, knowledge of details, keen eyes, generosity, curiosity, balancing of risks and rewards, planning, improvisation and knowing how to untie seemingly impossible knots have been essential for this expedition. Science is in many respects like any other adventure and it has also been a great pleasure to benefit from the same quali- ties with Kalle as a guide on many recreational tours on land, at sea, in snow and on ice as well as having countless inspiring lunch-talks about mountains waiting to be climbed. e collaboration with Professor Romas Kazlauskas’ group from McGill University, Montreal, Canada, has been very important. I have enjoyed learn- ing from the scientific expertise of Professor Kazlauskas and also received very valuable comments on the thesis manuscript. e chemical knowledge and intuition, the laboratory craftsmanship and the always good mood of Dr Seongsoon Park has been of great importance to me. Jari Alander at Karlshamns AB and Christian von Corswant and Erik Söderlind at AstraZeneca AB have been very important as experts in their respective fields and I have learned a lot by working with them as coauthors. e ideas and work of Katarina Ekelund and Per-Johan Lundberg have also been very valuable. e hospitality and generosity of all the group members, both at Karlshamns and AstraZeneca, have been very stimulating. All the past and present members in the Biocatalysis group have been es- sential for science, learning and many other fun things. Especially Professor Torbjörn Norin for ideas, discussions and advice both in the collaboration with the Biocatalysis group and within SNAP. anks also to all colleagues at the Department of Biotechnology for an enjoyable time. I would like to especially mention Tommy Östlund who has
62 63 Acknowledgements
built and fixed countless small but important laboratory things for me, and Alphonsa Lourdudoss and Lotta Rosenfeldt who have brought administrative order to a chaotic environment. Special thanks to my constant office-mate, lab-partner and time-keeper Jenny Ottosson who has been a reliable support in all situations. Per Berglund for movies, great skiing and for the photograph on page . Linda Fransson for Dajms, tea and great discussions. Joke Rotticci-Mulder and Didier Rotticci for all sorts of great ideas, skating and canoeing. Åsa Kallas for tea, thoughts and for playing squash – always fair, for fun and to win. Lena Gumaelius for always being positive. Jill Sigrell for laughs, salt water, sunshine and good talks. anks to Johan Håkans for many deep thoughts, for some not so deep and for some truly great cheesecakes. Bertil Johansson, Peter Sjödin and Johan for fantastic trips to Gotska Sandön and Skåne and for some really early morn- ings. Marie Ronnegård for much support. Johan and Malin Einarsson with family for their friendship. anks also to all Gamecorner- and Innebandy- players for deactivating my brain and activating my body, respectively. Finally I would like to thank my family. Erika, Henrik and Sofie for much joy and many great dinners. Carina for being there. My parents Anna-Lena and Harry for their endless love, support and for always encouraging me to discover.
62 63 References
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